RNAi Constructs for Inhibiting ASGR1 Expression and Methods of Use Thereof

ABSTRACT

The present invention relates to RNAi constructs for reducing expression of the ASGR1 gene. Methods of using such RNAi constructs to treat or prevent cardiovascular disease, such as coronary artery disease and myocardial infarction, and to reduce serum non-HDL cholesterol levels are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/328,221, filed Feb. 25, 2019, which is the national stage entry ofPCT Application No. PCT/US2017/048757, filed Aug. 25, 2017, which claimsthe benefit of U.S. Provisional Application No. 62/380,216, filed Aug.26, 2016, all of which are hereby incorporated by reference in theirentireties.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The present application contains a Sequence Listing, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The computer readable format copy of theSequence Listing, which was created on Nov. 13, 2020, is namedA-2094-US-CNT_SubSeq_ST25 and is 1.49 megabytes in size.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for modulatingliver expression of asialoglycoprotein receptor 1 (ASGR1). Inparticular, the present invention relates to nucleic acid-basedtherapeutics for reducing ASGR1 expression via RNA interference andmethods of using such nucleic acid-based therapeutics to treat orprevent cardiovascular disease.

BACKGROUND OF THE INVENTION

Despite the many advancements and new therapeutics that have emergedover the last several years, cardiovascular disease remains the leadingcause of death worldwide. One out of every three adults in America hassome form of cardiovascular disease, including coronary artery disease,myocardial infarction, angina, heart failure, and stroke (Heart diseaseand stroke statistics-2016 update: a report from the American HeartAssociation. Circulation, Vol. 133:e38-e360, 2016). In 2013, over 17.3million people globally and 1.4 million people in the United States diedfrom some form of cardiovascular disease, accounting for 31% of allglobal deaths and 54% of all deaths in the U.S. that year (Heart diseaseand stroke statistics-2016 update). Cardiovascular disease currentlyclaims more lives each year than the next two leading causes of death,cancer and chronic lower respiratory disease, combined (Heart diseaseand stroke statistics-2016 update). Thus, there remains a need foradditional therapeutic agents for the treatment of cardiovasculardisease.

The asialoglycoprotein receptor is a calcium-dependent receptorexpressed on the surface of hepatocytes that contributes to the removaland degradation of desialylated glycoproteins from the serum by bindingto ligands with terminal galactose and N-acetylgalactosamine residues(Weigel, Bioessays, Vol. 16:519-524, 1994; Stockert, Physiol. Rev., Vol.75: 591-609, 1995). The hetero-oligomeric asialoglycoprotein receptor iscomprised of two different proteins, a 48 kDa asialoglycoproteinreceptor 1 (ASGR1) major subunit and a 40 kDa asialoglycoproteinreceptor 2 (ASGR2) minor subunit (see, e.g., Stockert, 1995). Theasialogylcoprotein receptor has been implicated in the clearance of lowdensity lipoproteins and chylomicron remnants, suggesting a role for thereceptor in lipoprotein metabolism (Windier et al., Biochem J., Vol.276(Pt 1):79-87, 1991; Ishibashi et al., J Biol Chem., Vol.271:22422-22427, 1996). Recently, human carriers of loss of functionvariant alleles of the ASGR1 subunit of the asialoglycoprotein receptorwere reported to have lower serum levels of non-high-density lipoprotein(HDL) cholesterol and a lower risk of coronary artery disease andmyocardial infarction as compared to non-carriers (Nioi et al., NewEngland Journal of Medicine, Vol. 374(22):2131-2141, 2016). Accordingly,therapeutics targeting ASGR1 function represent a novel approach toreducing non-HDL cholesterol levels and treating cardiovascular disease,particularly coronary artery disease.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the design and generation ofRNAi constructs that target the ASGR1 gene and reduce expression ofASGR1 in liver cells. The sequence-specific inhibition of ASGR1expression is useful for treating or preventing conditions associatedwith ASGR1 expression, such as cardiovascular disease. Accordingly, inone embodiment, the present invention provides an RNAi constructcomprising a sense strand and an antisense strand, wherein the antisensestrand comprises a region having a sequence that is complementary to anASGR1 mRNA sequence. In certain embodiments, the antisense strandcomprises a region having at least 15 contiguous nucleotides from anantisense sequence listed in Table 1, Table 6, or Table 8.

In some embodiments, the sense strand of the RNAi constructs describedherein comprises a sequence that is sufficiently complementary to thesequence of the antisense strand to form a duplex region of about 15 toabout 30 base pairs in length. In these and other embodiments, the senseand antisense strands each are about 15 to about 30 nucleotides inlength. In some embodiments, the RNAi constructs comprise at least oneblunt end. In other embodiments, the RNAi constructs comprise at leastone nucleotide overhang. Such nucleotide overhangs may comprise 1 to 6unpaired nucleotides and can be located at the 3′ end of the sensestrand, the 3′ end of the antisense strand, or the 3′ end of both thesense and antisense strand. In certain embodiments, the RNAi constructscomprise an overhang of two unpaired nucleotides at the 3′ end of thesense strand and the 3′ end of the antisense strand. In otherembodiments, the RNAi constructs comprise an overhang of two unpairednucleotides at the 3′ end of the antisense strand and a blunt end of the3′ end of the sense strand/5′ end of the antisense strand.

The RNAi constructs of the invention may comprise one or more modifiednucleotides, including nucleotides having modifications to the ribosering, nucleobase, or phosphodiester backbone. In some embodiments, theRNAi constructs comprise one or more 2′-modified nucleotides. Such2′-modified nucleotides can include 2′-fluoro modified nucleotides,2′-O-methyl modified nucleotides, 2′-O-methoxyethyl modifiednucleotides, 2′-O-allyl modified nucleotides, bicyclic nucleic acids(BNA), or combinations thereof. In one particular embodiment, the RNAiconstructs comprise one or more 2′-fluoro modified nucleotides,2′-O-methyl modified nucleotides, or combinations thereof. In someembodiments, all of the nucleotides in the sense and antisense strand ofthe RNAi construct are modified nucleotides.

In some embodiments, the RNAi constructs comprise at least one backbonemodification, such as a modified internucleotide or internucleosidelinkage. In certain embodiments, the RNAi constructs described hereincomprise at least one phosphorothioate internucleotide linkage. Inparticular embodiments, the phosphorothioate internucleotide linkagesmay be positioned at the 3′ or 5′ ends of the sense and/or antisensestrands.

In some embodiments, the antisense strand and/or the sense strand of theRNAi constructs of the invention may comprise or consist of a sequencefrom the antisense and sense sequences listed in Tables 1, 6, or 8. Incertain embodiments, the RNAi construct may be any one of the duplexcompounds listed in any one of Tables 1 to 10. In one embodiment, theRNAi construct is D-1098, D-1176, D-1200, D-1206, D-1235, D-1246,D-1373, D-1389, D-1813, D-1815, D-1983, D-2000, D-2045, D-2142, D-2143,D-1438, D-1494, D-2357, D-2359, D-2361, D-2365, D-2461, D-3036, D-3037,D-3051, D-3053, D-3057, D-3779, D-3780, D-3782, D-3788, D-3791, D-3795,D-3799, or D-3800. In another embodiment, the RNAi construct is D-1200,D-1206, D-1235, D-1815, D-2143, D-2359, D-2361, D-2365, D-2142, D-1176,D-3779, D-3782, D-3788, D-3799, or D-3800. In another embodiment, theRNAi construct is D-2359. In another embodiment, the RNAi construct isD-1815. In yet another embodiment, the RNAi construct is D-1235. Instill another embodiment, the RNAi construct is D-2143. In anotherembodiment, the RNAi construct is D-2361. In some embodiments, the RNAiconstruct is D-3782. In other embodiments, the RNAi construct is D-3799.

The RNAi constructs may further comprise a ligand to facilitate deliveryor uptake of the RNAi constructs to specific tissues or cells, such asliver cells. In certain embodiments, the ligand targets delivery of theRNAi constructs to hepatocytes. In these and other embodiments, theligand may comprise galactose, galactosamine, or N-acetyl-galactosamine(GalNAc). In certain embodiments, the ligand comprises a multivalentgalactose or multivalent GalNAc moiety, such as a trivalent ortetravalent galactose or GalNAc moiety. The ligand may be covalentlyattached to the 5′ or 3′ end of the sense strand of the RNAi construct,optionally through a linker. In some embodiments, the RNAi constructscomprise a ligand and linker having a structure according to any ofFormulas I to XXIX described herein. In certain embodiments, the RNAiconstructs comprise a ligand and linker having a structure according toFormula VII, Formula VIII, Formula XVI, Formula XXVI, or Formula XXIX.In one embodiment, the RNAi constructs comprise a ligand and linkerhaving a structure according to Formula XVI, wherein n=1 and k=3.

In certain embodiments, the ligand may comprise an antibody orantigen-binding fragment thereof that specifically binds to ASGR1. The5′ or 3′ end of the sense strand of the RNAi construct may be covalentlylinked to the antibody or antigen-binding fragment through the sidechain of an amino acid residue in the light chain or heavy chain of theantibody or antigen-binding fragment. In some embodiments, the sensestrand of the RNAi construct is covalently attached, optionally througha linker, to the side chain of a cysteine residue present in the heavychain or light chain of the antibody or antigen-binding fragmentthereof. In one embodiment, the anti-ASGR1 antibody-RNA moleculeconjugate comprises at least one copy of the interfering RNA molecule(e.g. siRNA or shRNA). In another embodiment, the anti-ASGR1antibody-RNA molecule conjugate comprises two copies of the interferingRNA molecule (e.g. siRNA or shRNA).

The present invention also provides pharmaceutical compositionscomprising any of the RNAi constructs described herein and apharmaceutically acceptable carrier, excipient, or diluent. Suchpharmaceutical compositions are particularly useful for reducingexpression of ASGR1 in the cells (e.g. liver cells) of a patient in needthereof. Patients who may be administered a pharmaceutical compositionof the invention can include patients with a history of myocardialinfarction, patients diagnosed with or at risk for coronary arterydisease or other form of cardiovascular disease, and patients withelevated levels of non-HDL cholesterol. Accordingly, the presentinvention includes methods of treating or preventing cardiovasculardisease in a patient in need thereof by administering an RNAi constructor pharmaceutical composition described herein. In certain embodiments,the present invention provides methods for reducing non-HDL cholesterolin a patient in need thereof by administering an RNAi construct orpharmaceutical composition described herein.

The use of ASGR1-targeting RNAi constructs in any of the methodsdescribed herein or for preparation of medicaments for administrationaccording to the methods described herein is specifically contemplated.For instance, the present invention includes an ASGR1-targeting RNAiconstruct for use in a method for treating or preventing cardiovasculardisease, including coronary artery disease or myocardial infarction, ina patient in need thereof. The present invention also includes anASGR1-targeting RNAi construct for use in a method for reducing non-HDLcholesterol in a patient in need thereof. In some embodiments, thepresent invention provides an ASGR1-targeting RNAi construct for use ina method for reducing the risk of myocardial infarction in a patient inneed thereof.

The present invention also encompasses the use of an ASGR1-targetingRNAi construct in the preparation of a medicament for treating orpreventing cardiovascular disease, including coronary artery disease ormyocardial infarction, in a patient in need thereof. In certainembodiments, the present invention provides the use of anASGR1-targeting RNAi construct in the preparation of a medicament forreducing non-HDL cholesterol in a patient in need thereof. In certainother embodiments, the present invention provides the use of anASGR1-targeting RNAi construct in the preparation of a medicament forreducing the risk of myocardial infarction in a patient in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleotide sequence of transcript variant 1 of humanASGR1 (NCBI Reference Sequence No. NM_001671.4; SEQ ID NO: 1). Thetranscript sequence is depicted as the complementary DNA (cDNA) sequencewith thymine bases replacing uracil bases.

FIG. 1B shows the nucleotide sequence of transcript variant 2 of humanASGR1 (NCBI Reference Sequence No. NM_001197216.2; SEQ ID NO: 2). Thetranscript sequence is depicted as the cDNA sequence with thymine basesreplacing uracil bases.

FIG. 2 shows the nucleotide sequence of transcript variant 1 of mouseAsgr1 (NCBI Reference Sequence No. NM_009714.2; SEQ ID NO: 3011). Thetranscript sequence is depicted as the complementary DNA (cDNA) sequencewith thymine bases replacing uracil bases.

FIG. 3 shows the nucleotide sequence of a transcript of rat Asgr1 (SEQID NO: 3012). The transcript sequence is depicted as the complementaryDNA (cDNA) sequence with thymine bases replacing uracil bases.

FIG. 4 shows the nucleotide sequence of a transcript of macaque (Macacafascicularis) ASGR1 (NCBI Reference Sequence No. XM_005582698.1; SEQ IDNO: 3013). The transcript sequence is depicted as the complementary DNA(cDNA) sequence with thymine bases replacing uracil bases.

FIG. 5 depicts the synthetic scheme for a tetravalent GalNAc moiety thatcan be incorporated into any of the RNAi constructs of the invention.

FIG. 6A is a bar graph of human ASGR1 expression levels in livers ofASGR1 knockout mice injected with an AAV encoding human ASGR1 andtreated with 5 mg/kg subcutaneous injections of the indicatedGalNAc-ASGR1 siRNA conjugates. Human ASGR1 expression was measured byqPCR and is reported as expression levels relative to the AAV onlycontrol animals, which were ASGR1 knockout animals injected with the AAVencoding human ASGR1, but were otherwise untreated. Expression levelsare shown at day 8 (d8) and day 15 (d15) after GalNAc-siRNA conjugateadministration. Wild-type (WT) mice and ASGR1 knockout (KO) animals wereincluded as controls.

FIG. 6B is a bar graph of serum levels of alkaline phosphatase (ALP)from the animals described in FIG. 6A. Serum was obtained at day 8 (d8)and day 15 (d15) following administration of the indicated GalNAc-siRNAconjugates.

FIG. 7A is a schematic illustrating the reaction to add a bromoacetyllinker to the 3′ end of the sense strand of a siRNA duplex.

FIG. 7B is a schematic depicting the conjugation reaction to attach asiRNA duplex to an anti-ASGR1 antibody.

FIG. 8 is a bar graph showing a dose-dependent knockdown of ASGR1 mRNAin human primary hepatocytes observed with the 3549 (RNA-Ab ratio of 1)and 3550 (RNA-Ab ratio of 2) anti-ASGR1 mAb-siRNA conjugates. Theunconjugated anti-ASGR1 cys mAb (PL-53515) was used as a control.

FIG. 9A is a bar graph showing the ASGR1 mRNA level in livers fromwild-type mice in all dosing groups measured at the indicated timepoints (days 2, 4, 8, and 15). The same siRNA conjugated to a GalNAcmoiety (compound 1418) was used as a positive control. The amount ofsiRNA in 5 mpk of 1418 is equivalent to that in 30 mpk of the 3550compound, which has 2 siRNAs/mAb.

FIG. 9B is a line graph depicting ASGR1 protein expression in liversfrom wild-type mice in all dosing groups measured at the indicated timepoints (days 2, 4, 8, and 15). The same siRNA conjugated to a GalNAcmoiety (compound 1418) was used as a positive control. The amount ofsiRNA in 5 mpk of 1418 is equivalent to that in 30 mpk of the 3550compound, which has 2 siRNAs/mAb.

FIG. 10 is a bar graph showing serum alkaline phosphatase (ALP) fromwild-type mice in all dosing groups measured at the indicated timepoints (days 2, 4, 8, and 15). The same siRNA conjugated to a GalNAcmoiety (compound 1418) was used as a positive control. The amount ofsiRNA in 5 mpk of 1418 is equivalent to that in 30 mpk of the 3550compound, which has 2 siRNAs/mAb.

DETAILED DESCRIPTION

The present invention is directed to compositions and methods forregulating the expression of the asialoglycoprotein receptor in a cellor mammal. In some embodiments, compositions of the invention compriseRNAi constructs that target an ASGR1 mRNA and reduce ASGR1 expression ina cell or mammal. Such RNAi constructs are useful for treating orpreventing various forms of cardiovascular disease, such as, forexample, by reducing non-HDL cholesterol serum levels and reducing therisk of developing coronary artery disease or myocardial infarction.

As used herein, the term “RNAi construct” refers to an agent comprisinga RNA molecule that is capable of downregulating expression of a targetgene (e.g. ASGR1) via a RNA interference mechanism when introduced intoa cell. RNA interference is the process by which a nucleic acid moleculeinduces the cleavage and degradation of a target RNA molecule (e.g.messenger RNA or mRNA molecule) in a sequence-specific manner, e.g.through a RNA-induced silencing complex (RISC) pathway. In someembodiments, the RNAi construct comprises a double-stranded RNA moleculecomprising two antiparallel strands of contiguous nucleotides that aresufficiently complementary to each other to hybridize to form a duplexregion. “Hybridize” or “hybridization” refers to the pairing ofcomplementary polynucleotides, typically via hydrogen bonding (e.g.Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) betweencomplementary bases in the two polynucleotides. The strand comprising aregion having a sequence that is substantially complementary to a targetsequence (e.g. target mRNA) is referred to as the “antisense strand.”The “sense strand” refers to the strand that includes a region that issubstantially complementary to a region of the antisense strand. In someembodiments, the sense strand may comprise a region that has a sequencethat is substantially identical to the target sequence.

A double-stranded RNA molecule may include chemical modifications toribonucleotides, including modifications to the ribose sugar, base, orbackbone components of the ribonucleotides, such as those describedherein or known in the art. Any such modifications, as used in adouble-stranded RNA molecule (e.g. siRNA, shRNA, or the like), areencompassed by the term “double-stranded RNA” for the purposes of thisdisclosure.

As used herein, a first sequence is “complementary” to a second sequenceif a polynucleotide comprising the first sequence can hybridize to apolynucleotide comprising the second sequence to form a duplex regionunder certain conditions, such as physiological conditions. Other suchconditions can include moderate or stringent hybridization conditions,which are known to those of skill in the art. A first sequence isconsidered to be fully complementary (100% complementary) to a secondsequence if a polynucleotide comprising the first sequence base pairswith a polynucleotide comprising the second sequence over the entirelength of one or both nucleotide sequences without any mismatches. Asequence is “substantially complementary” to a target sequence if thesequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%complementary to a target sequence. Percent complementarity can becalculated by dividing the number of bases in a first sequence that arecomplementary to bases at corresponding positions in a second or targetsequence by the total length of the first sequence. A sequence may alsobe said to be substantially complementary to another sequence if thereare no more than 5, 4, 3, or 2 mismatches over a 30 base pair duplexregion when the two sequences are hybridized. Generally, if anynucleotide overhangs, as defined herein, are present, the sequence ofsuch overhangs is not considered in determining the degree ofcomplementarity between two sequences. By way of example, a sense strandof 21 nucleotides in length and an antisense strand of 21 nucleotides inlength that hybridize to form a 19 base pair duplex region with a 2nucleotide overhang at the 3′ end of each strand would be considered tobe fully complementary as the term is used herein.

In some embodiments, a region of the antisense strand comprises asequence that is fully complementary to a region of the target RNAsequence (e.g. ASGR1 mRNA). In such embodiments, the sense strand maycomprise a sequence that is fully complementary to the sequence of theantisense strand. In other such embodiments, the sense strand maycomprise a sequence that is substantially complementary to the sequenceof the antisense strand, e.g. having 1, 2, 3, 4, or 5 mismatches in theduplex region formed by the sense and antisense strands. In certainembodiments, it is preferred that any mismatches occur within theterminal regions (e.g. within 6, 5, 4, 3, or 2 nucleotides of the 5′and/or 3′ ends of the strands). In one embodiment, any mismatches in theduplex region formed from the sense and antisense strands occur within6, 5, 4, 3, or 2 nucleotides of the 5′ end of the antisense strand.

In certain embodiments, the sense strand and antisense strand of thedouble-stranded RNA may be two separate molecules that hybridize to forma duplex region, but are otherwise unconnected. Such double-stranded RNAmolecules formed from two separate strands are referred to as “smallinterfering RNAs” or “short interfering RNAs” (siRNAs). Thus, in someembodiments, the RNAi constructs of the invention comprise a siRNA.

In other embodiments, the sense strand and the antisense strand thathybridize to form a duplex region may be part of a single RNA molecule,i.e. the sense and antisense strands are part of a self-complementaryregion of a single RNA molecule. In such cases, a single RNA moleculecomprises a duplex region (also referred to as a stem region) and a loopregion. The 3′ end of the sense strand is connected to the 5′ end of theantisense strand by a contiguous sequence of unpaired nucleotides, whichwill form the loop region. The loop region is typically of a sufficientlength to allow the RNA molecule to fold back on itself such that theantisense strand can base pair with the sense strand to form the duplexor stem region. The loop region can comprise from about 3 to about 25,from about 5 to about 15, or from about 8 to about 12 unpairednucleotides. Such RNA molecules with at least partiallyself-complementary regions are referred to as “short hairpin RNAs”(shRNAs). In certain embodiments, the RNAi constructs of the inventioncomprise a shRNA. The length of a single, at least partiallyself-complementary RNA molecule can be from about 35 nucleotides toabout 100 nucleotides, from about 45 nucleotides to about 85nucleotides, or from about 50 to about 60 nucleotides and comprise aduplex region and loop region each having the lengths recited herein.

In some embodiments, the RNAi constructs of the invention comprise asense strand and an antisense strand, wherein the antisense strandcomprises a region having a sequence that is substantially or fullycomplementary to an ASGR1 messenger RNA (mRNA) sequence. As used herein,an “ASGR1 mRNA sequence” refers to any messenger RNA sequence, includingsplice variants, encoding an ASGR1 protein, including ASGR1 proteinvariants or isoforms from any species (e.g. mouse, rat, non-humanprimate, human). ASGR1 protein (also known as HL-1, ASGPR H1, ASGPR1,and CLEC4H1), as used herein, refers to the major subunit of theasialoglycoprotein receptor. In humans, ASGR1 is found on chromosome17p13.2 and is expressed as two different isoforms, a long isoform (H1aor isoform A) of about 291 amino acids and a short soluble isoform (H1bor isoform B) of about 252 amino acids.

An ASGR1 mRNA sequence also includes the transcript sequence expressedas its complementary DNA (cDNA) sequence. A cDNA sequence refers to thesequence of an mRNA transcript expressed as DNA bases (e.g. guanine,adenine, thymine, and cytosine) rather than RNA bases (e.g. guanine,adenine, uracil, and cytosine). Thus, the antisense strand of the RNAiconstructs of the invention may comprise a region having a sequence thatis substantially or fully complementary to a target ASGR1 mRNA sequenceor ASGR1 cDNA sequence. An ASGR1 mRNA or cDNA sequence can include, butis not limited to, any ASGR1 mRNA or cDNA sequence selected from theNCBI Reference sequences NM_001671.4 (human; FIG. 1A, SEQ ID NO: 1),NM_001197216.2 (human; FIG. 1B, SEQ ID NO: 2), NM_009714.2 (mouse; FIG.2, SEQ ID NO: 3011), and XM_005582698.1 (cynomolgus monkey; FIG. 4, SEQID NO: 3013) or the rat sequence in FIG. 3 (SEQ ID NO: 3012). In oneembodiment, the ASGR1 mRNA sequence is human transcript variant 1 listedin the NCBI database as Reference Sequence NM_001671.4 (see FIG. 1A; SEQID NO: 1). In another embodiment, the ASGR1 mRNA sequence is humantranscript variant 2 listed in the NCBI database as Reference SequenceNM_001197216.2 (see FIG. 1B; SEQ ID NO: 2).

A region of the antisense strand can be substantially complementary orfully complementary to at least 15 consecutive nucleotides of the ASGR1mRNA sequence. In some embodiments, the target region of the ASGR1 mRNAsequence to which the antisense strand comprises a region ofcomplementarity can range from about 15 to about 30 consecutivenucleotides, from about 16 to about 28 consecutive nucleotides, fromabout 18 to about 26 consecutive nucleotides, from about 17 to about 24consecutive nucleotides, from about 19 to about 25 consecutivenucleotides, from about 19 to about 23 consecutive nucleotides, or fromabout 19 to about 21 consecutive nucleotides. In certain embodiments,the region of the antisense strand comprising a sequence that issubstantially or fully complementary to an ASGR1 mRNA sequence may, insome embodiments, comprise at least 15 contiguous nucleotides from anantisense sequence listed in Table 1, Table 6, or Table 8. In otherembodiments, the antisense sequence comprises at least 16, at least 17,at least 18, or at least 19 contiguous nucleotides from an antisensesequence listed in Table 1, Table 6, or Table 8. For instance, in someembodiments, the region of the antisense strand comprising a sequencethat is substantially or fully complementary to an ASGR1 mRNA sequencecomprises at least 15 contiguous nucleotides from a sequence selectedfrom SEQ ID NO: 1606, SEQ ID NO: 1684, SEQ ID NO: 1708, SEQ ID NO: 1714,SEQ ID NO: 1743, SEQ ID NO: 1754, SEQ ID NO: 1881, SEQ ID NO: 1897, SEQID NO: 2321, SEQ ID NO: 2323, SEQ ID NO: 2491, SEQ ID NO: 2508, SEQ IDNO: 2553, SEQ ID NO: 2650, SEQ ID NO: 2651, SEQ ID NO: 1946, SEQ ID NO:2002, SEQ ID NO: 2865, SEQ ID NO: 2867, SEQ ID NO: 2869, SEQ ID NO:2873, SEQ ID NO: 2969, SEQ ID NO: 3701, SEQ ID NO: 3702, SEQ ID NO:3716, SEQ ID NO: 3718, SEQ ID NO: 3722, SEQ ID NO: 4618, SEQ ID NO:4619, SEQ ID NO: 4621, SEQ ID NO: 4627, SEQ ID NO: 4630, SEQ ID NO:4634, SEQ ID NO: 4638, or SEQ ID NO: 4639.

The sense strand of the RNAi construct typically comprises a sequencethat is sufficiently complementary to the sequence of the antisensestrand such that the two strands hybridize under physiologicalconditions to form a duplex region. A “duplex region” refers to theregion in two complementary or substantially complementarypolynucleotides that form base pairs with one another, either byWatson-Crick base pairing or other hydrogen bonding interaction, tocreate a duplex between the two polynucleotides. The duplex region ofthe RNAi construct should be of sufficient length to allow the RNAiconstruct to enter the RNA interference pathway, e.g. by engaging theDicer enzyme and/or the RISC complex. For instance, in some embodiments,the duplex region is about 15 to about 30 base pairs in length. Otherlengths for the duplex region within this range are also suitable, suchas about 15 to about 28 base pairs, about 15 to about 26 base pairs,about 15 to about 24 base pairs, about 15 to about 22 base pairs, about17 to about 28 base pairs, about 17 to about 26 base pairs, about 17 toabout 24 base pairs, about 17 to about 23 base pairs, about 17 to about21 base pairs, about 19 to about 25 base pairs, about 19 to about 23base pairs, or about 19 to about 21 base pairs. In one embodiment, theduplex region is about 17 to about 24 base pairs in length. In anotherembodiment, the duplex region is about 19 to about 21 base pairs inlength.

For embodiments in which the sense strand and antisense strand are twoseparate molecules (e.g. RNAi construct comprises a siRNA), the sensestrand and antisense strand need not be the same length as the length ofthe duplex region. For instance, one or both strands may be longer thanthe duplex region and have one or more unpaired nucleotides ormismatches flanking the duplex region. Thus, in some embodiments, theRNAi construct comprises at least one nucleotide overhang. As usedherein, a “nucleotide overhang” refers to the unpaired nucleotide ornucleotides that extend beyond the duplex region at the terminal ends ofthe strands. Nucleotide overhangs are typically created when the 3′ endof one strand extends beyond the 5′ end of the other strand or when the5′ end of one strand extends beyond the 3′ end of the other strand. Thelength of a nucleotide overhang is generally between 1 and 6nucleotides, 1 and 5 nucleotides, 1 and 4 nucleotides, 1 and 3nucleotides, 2 and 6 nucleotides, 2 and 5 nucleotides, or 2 and 4nucleotides. In some embodiments, the nucleotide overhang comprises 1,2, 3, 4, 5, or 6 nucleotides. In one particular embodiment, thenucleotide overhang comprises 1 to 4 nucleotides. In certainembodiments, the nucleotide overhang comprises 2 nucleotides. Thenucleotides in the overhang can be ribonucleotides,deoxyribonucleotides, or modified nucleotides as described herein. Insome embodiments, the overhang comprises a 5′-uridine-uridine-3′(5′-UU-3′) dinucleotide. In such embodiments, the UU dinucleotide maycomprise ribonucleotides or modified nucleotides, e.g. 2′-modifiednucleotides. In other embodiments, the overhang comprises a5′-deoxythymidine-deoxythymidine-3′ (5′-dTdT-3′) dinucleotide.

The nucleotide overhang can be at the 5′ end or 3′ end of one or bothstrands. For example, in one embodiment, the RNAi construct comprises anucleotide overhang at the 5′ end and the 3′ end of the antisensestrand. In another embodiment, the RNAi construct comprises a nucleotideoverhang at the 5′ end and the 3′ end of the sense strand. In someembodiments, the RNAi construct comprises a nucleotide overhang at the5′ end of the sense strand and the 5′ end of the antisense strand. Inother embodiments, the RNAi construct comprises a nucleotide overhang atthe 3′ end of the sense strand and the 3′ end of the antisense strand.

The RNAi constructs may comprise a single nucleotide overhang at one endof the double-stranded RNA molecule and a blunt end at the other. A“blunt end” means that the sense strand and antisense strand are fullybase-paired at the end of the molecule and there are no unpairednucleotides that extend beyond the duplex region. In some embodiments,the RNAi construct comprises a nucleotide overhang at the 3′ end of thesense strand and a blunt end at the 5′ end of the sense strand and 3′end of the antisense strand. In other embodiments, the RNAi constructcomprises a nucleotide overhang at the 3′ end of the antisense strandand a blunt end at the 5′ end of the antisense strand and the 3′ end ofthe sense strand. In certain embodiments, the RNAi construct comprises ablunt end at both ends of the double-stranded RNA molecule. In suchembodiments, the sense strand and antisense strand have the same lengthand the duplex region is the same length as the sense and antisensestrands (i.e. the molecule is double-stranded over its entire length).

The sense strand and antisense strand can each independently be about 15to about 30 nucleotides in length, about 18 to about 28 nucleotides inlength, about 19 to about 27 nucleotides in length, about 19 to about 25nucleotides in length, about 19 to about 23 nucleotides in length, about21 to about 25 nucleotides in length, or about 21 to about 23nucleotides in length. In certain embodiments, the sense strand andantisense strand are each about 18, about 19, about 20, about 21, about22, about 23, about 24, or about 25 nucleotides in length. In someembodiments, the sense strand and antisense strand have the same lengthbut form a duplex region that is shorter than the strands such that theRNAi construct has two nucleotide overhangs. For instance, in oneembodiment, the RNAi construct comprises (i) a sense strand and anantisense strand that are each 21 nucleotides in length, (ii) a duplexregion that is 19 base pairs in length, and (iii) nucleotide overhangsof 2 unpaired nucleotides at both the 3′ end of the sense strand and the3′ end of the antisense strand. In another embodiment, the RNAiconstruct comprises (i) a sense strand and an antisense strand that areeach 23 nucleotides in length, (ii) a duplex region that is 21 basepairs in length, and (iii) nucleotide overhangs of 2 unpairednucleotides at both the 3′ end of the sense strand and the 3′ end of theantisense strand. In other embodiments, the sense strand and antisensestrand have the same length and form a duplex region over their entirelength such that there are no nucleotide overhangs on either end of thedouble-stranded molecule. In one such embodiment, the RNAi construct isblunt ended and comprises (i) a sense strand and an antisense strand,each of which is 21 nucleotides in length, and (ii) a duplex region thatis 21 base pairs in length. In another such embodiment, the RNAiconstruct is blunt ended and comprises (i) a sense strand and anantisense strand, each of which is 23 nucleotides in length, and (ii) aduplex region that is 23 base pairs in length.

In other embodiments, the sense strand or the antisense strand is longerthan the other strand and the two strands form a duplex region having alength equal to that of the shorter strand such that the RNAi constructcomprises at least one nucleotide overhang. For example, in oneembodiment, the RNAi construct comprises (i) a sense strand that is 19nucleotides in length, (ii) an antisense strand that is 21 nucleotidesin length, (iii) a duplex region of 19 base pairs in length, and (iv) asingle nucleotide overhang of 2 unpaired nucleotides at the 3′ end ofthe antisense strand. In another embodiment, the RNAi constructcomprises (i) a sense strand that is 21 nucleotides in length, (ii) anantisense strand that is 23 nucleotides in length, (iii) a duplex regionof 21 base pairs in length, and (iv) a single nucleotide overhang of 2unpaired nucleotides at the 3′ end of the antisense strand.

The antisense strand of the RNAi constructs of the invention cancomprise the sequence of any one of the antisense sequences listed inTable 1, Table 6, or Table 8, the sequence of nucleotides 1-19 of any ofthese antisense sequences, or the sequence of nucleotides 2-19 of any ofthese antisense sequences. Each of the antisense sequences listed inTables 1, 6, and 8 comprises a sequence of at least 19 consecutivenucleotides (first 19 nucleotides counting from the 5′ end) that iscomplementary to an ASGR1 mRNA sequence plus a two nucleotide overhangsequence. Thus, in some embodiments, the antisense strand comprises asequence of nucleotides 1-19 of any one of SEQ ID NOs: 1508-3010,3665-4315, or 4513-4687. In other embodiments, the antisense strandcomprises a sequence of nucleotides 2-19 of any one of SEQ ID NOs:1508-3010, 3665-4315, or 4513-4687. In still other embodiments, theantisense strand comprises a sequence selected from SEQ ID NOs:1508-3010, 3665-4315, or 4513-4687. In certain embodiments, theantisense strand comprises, or consists of, a sequence selected from SEQID NO: 1606, SEQ ID NO: 1684, SEQ ID NO: 1708, SEQ ID NO: 1714, SEQ IDNO: 1743, SEQ ID NO: 1754, SEQ ID NO: 1881, SEQ ID NO: 1897, SEQ ID NO:2321, SEQ ID NO: 2323, SEQ ID NO: 2491, SEQ ID NO: 2508, SEQ ID NO:2553, SEQ ID NO: 2650, SEQ ID NO: 2651, SEQ ID NO: 1946, SEQ ID NO:2002, SEQ ID NO: 2865, SEQ ID NO: 2867, SEQ ID NO: 2869, SEQ ID NO:2873, SEQ ID NO: 2969, SEQ ID NO: 3701, SEQ ID NO: 3702, SEQ ID NO:3716, SEQ ID NO: 3718, SEQ ID NO: 3722, SEQ ID NO: 4618, SEQ ID NO:4619, SEQ ID NO: 4621, SEQ ID NO: 4627, SEQ ID NO: 4630, SEQ ID NO:4634, SEQ ID NO: 4638, or SEQ ID NO: 4639. In some embodiments, theantisense strand comprises, or consists of, a sequence selected from SEQID NO: 1684, SEQ ID NO: 1708, SEQ ID NO: 1714, SEQ ID NO: 1743, SEQ IDNO: 2323, SEQ ID NO: 2650, SEQ ID NO: 2651, SEQ ID NO: 2867, SEQ ID NO:2869, SEQ ID NO: 2873, SEQ ID NO: 4618, SEQ ID NO: 4621, SEQ ID NO:4627, SEQ ID NO: 4638, or SEQ ID NO: 4639. In other embodiments, theantisense strand comprises, or consists of, a sequence selected from SEQID NO: 1743, SEQ ID NO: 2323, SEQ ID NO: 2651, SEQ ID NO: 2867, SEQ IDNO: 2869, SEQ ID NO: 4621, or SEQ ID NO: 4638.

In these and other embodiments, the sense strand of the RNAi constructsof the invention can comprise the sequence of any one of the sensesequences listed in Table 1, Table 6, or Table 8, the sequence ofnucleotides 1-19 of any of these sense sequences, or the sequence ofnucleotides 2-19 of any of these sense sequences. Each of the sensesequences listed in Tables 1, 6, and 8 comprises a sequence of at least19 consecutive nucleotides (first 19 nucleotides counting from the 5′end) that is identical to an ASGR1 mRNA sequence and complementary tothe corresponding antisense sequence, plus a two nucleotide overhangsequence. Thus, in some embodiments, the sense strand comprises asequence of nucleotides 1-19 of any one of SEQ ID NOs: 5-1507,3014-3664, or 4319-4512. In other embodiments, the sense strandcomprises a sequence of nucleotides 2-19 of any one of SEQ ID NOs:5-1507, 3014-3664, or 4319-4512. In still other embodiments, the sensestrand comprises a sequence selected from SEQ ID NOs: 5-1507, 3014-3664,or 4319-4512. In certain embodiments, the sense strand comprises, orconsists of, a sequence selected from SEQ ID NO: 103, SEQ ID NO: 181,SEQ ID NO: 205, SEQ ID NO: 211, SEQ ID NO: 240, SEQ ID NO: 251, SEQ IDNO: 378, SEQ ID NO: 394, SEQ ID NO: 818, SEQ ID NO: 820, SEQ ID NO: 988,SEQ ID NO: 1005, SEQ ID NO: 1050, SEQ ID NO: 1147, SEQ ID NO: 1148, SEQID NO: 443, SEQ ID NO: 499, SEQ ID NO: 1362, SEQ ID NO 1364, SEQ ID NO:1366, SEQ ID NO: 1370, SEQ ID NO: 1466, SEQ ID NO: 3050, SEQ ID NO:3051, SEQ ID NO: 3065, SEQ ID NO: 3067, SEQ ID NO: 3071, SEQ ID NO:4443, SEQ ID NO: 4444, SEQ ID NO: 4446, SEQ ID NO: 4452, SEQ ID NO:4455, SEQ ID NO: 4459, SEQ ID NO: 4463, or SEQ ID NO: 4464. In certainother embodiments, the sense strand comprises, or consists of, asequence selected from SEQ ID NO: 181, SEQ ID NO: 205, SEQ ID NO: 211,SEQ ID NO: 240, SEQ ID NO: 820, SEQ ID NO: 1147, SEQ ID NO: 1148, SEQ IDNO: 1364, SEQ ID NO: 1366, SEQ ID NO: 1370, SEQ ID NO: 4443, SEQ ID NO:4446, SEQ ID NO: 4452, SEQ ID NO: 4463, or SEQ ID NO: 4464. In someembodiments, the sense strand comprises, or consists of, a sequenceselected from SEQ ID NO: 240, SEQ ID NO: 820, SEQ ID NO: 1148, SEQ IDNO: 1364, SEQ ID NO: 1366, SEQ ID NO: 4446 or SEQ ID NO: 4463.

In certain embodiments of the invention, the RNAi constructs comprise(i) a sense strand comprising a sequence selected from SEQ ID NOs:5-1507, 3014-3664, or 4319-4512, nucleotides 1-19 of any one of SEQ IDNOs: 5-1507, 3014-3664, or 4319-4512, or nucleotides 2-19 of any one ofSEQ ID NOs: 5-1507, 3014-3664, or 4319-4512, and (ii) an antisensestrand comprising a sequence selected from SEQ ID NOs: 1508-3010,3665-4315, or 4513-4687, nucleotides 1-19 of any one of SEQ ID NOs:1508-3010, 3665-4315, or 4513-4687, or nucleotides 2-19 of any one ofSEQ ID NOs: 1508-3010, 3665-4315, or 4513-4687. In some embodiments, theRNAi constructs comprise (i) a sense strand comprising, or consistingof, a sequence selected from SEQ ID NO: 103, SEQ ID NO: 181, SEQ ID NO:205, SEQ ID NO: 211, SEQ ID NO: 240, SEQ ID NO: 251, SEQ ID NO: 378, SEQID NO: 394, SEQ ID NO: 818, SEQ ID NO: 820, SEQ ID NO: 988, SEQ ID NO:1005, SEQ ID NO: 1050, SEQ ID NO: 1147, SEQ ID NO: 1148, SEQ ID NO: 443,SEQ ID NO: 499, SEQ ID NO: 1362, SEQ ID NO 1364, SEQ ID NO: 1366, SEQ IDNO: 1370, SEQ ID NO: 1466, SEQ ID NO: 3050, SEQ ID NO: 3051, SEQ ID NO:3065, SEQ ID NO: 3067, SEQ ID NO: 3071, SEQ ID NO: 4443, SEQ ID NO:4444, SEQ ID NO: 4446, SEQ ID NO: 4452, SEQ ID NO: 4455, SEQ ID NO:4459, SEQ ID NO: 4463, or SEQ ID NO: 4464 and (ii) an antisense strandcomprising, or consisting of, a sequence selected from SEQ ID NO: 1606,SEQ ID NO: 1684, SEQ ID NO: 1708, SEQ ID NO: 1714, SEQ ID NO: 1743, SEQID NO: 1754, SEQ ID NO: 1881, SEQ ID NO: 1897, SEQ ID NO: 2321, SEQ IDNO: 2323, SEQ ID NO: 2491, SEQ ID NO: 2508, SEQ ID NO: 2553, SEQ ID NO:2650, SEQ ID NO: 2651, SEQ ID NO: 1946, SEQ ID NO: 2002, SEQ ID NO:2865, SEQ ID NO: 2867, SEQ ID NO: 2869, SEQ ID NO: 2873, SEQ ID NO:2969, SEQ ID NO: 3701, SEQ ID NO: 3702, SEQ ID NO: 3716, SEQ ID NO:3718, SEQ ID NO: 3722, SEQ ID NO: 4618, SEQ ID NO: 4619, SEQ ID NO:4621, SEQ ID NO: 4627, SEQ ID NO: 4630, SEQ ID NO: 4634, SEQ ID NO:4638, or SEQ ID NO: 4639. In other embodiments, the RNAi constructscomprise (i) a sense strand comprising, or consisting of, a sequenceselected from SEQ ID NO: 181, SEQ ID NO: 205, SEQ ID NO: 211, SEQ ID NO:240, SEQ ID NO: 820, SEQ ID NO: 1147, SEQ ID NO: 1148, SEQ ID NO: 1364,SEQ ID NO: 1366, SEQ ID NO: 1370, SEQ ID NO: 4443, SEQ ID NO: 4446, SEQID NO: 4452, SEQ ID NO: 4463, or SEQ ID NO: 4464, and (ii) an antisensestrand comprising, or consisting of, a sequence selected from SEQ ID NO:1684, SEQ ID NO: 1708, SEQ ID NO: 1714, SEQ ID NO: 1743, SEQ ID NO:2323, SEQ ID NO: 2650, SEQ ID NO: 2651, SEQ ID NO: 2867, SEQ ID NO:2869, SEQ ID NO: 2873, SEQ ID NO: 4618, SEQ ID NO: 4621, SEQ ID NO:4627, SEQ ID NO: 4638, or SEQ ID NO: 4639. In still other embodiments,the RNAi constructs comprise (i) a sense strand comprising, orconsisting of, a sequence selected from SEQ ID NO: 240, SEQ ID NO: 820,SEQ ID NO: 1148, SEQ ID NO: 1364, SEQ ID NO: 1366, SEQ ID NO: 4446 orSEQ ID NO: 4463, and (ii) an antisense strand comprising, or consistingof, a sequence selected from SEQ ID NO: 1743, SEQ ID NO: 2323, SEQ IDNO: 2651, SEQ ID NO: 2867, SEQ ID NO: 2869, SEQ ID NO: 4621, or SEQ IDNO: 4638.

In certain embodiments, the RNAi constructs comprise: (a) a sense strandcomprising the sequence of SEQ ID NO: 181 and an antisense strandcomprising the sequence of SEQ ID NO: 1684; (b) a sense strandcomprising the sequence of SEQ ID NO: 205 and an antisense strandcomprising the sequence of SEQ ID NO: 1708; (c) a sense strandcomprising the sequence of SEQ ID NO: 211 and an antisense strandcomprising the sequence of SEQ ID NO: 1714; (d) a sense strandcomprising the sequence of SEQ ID NO: 240 and an antisense strandcomprising the sequence of SEQ ID NO: 1743; (e) a sense strandcomprising the sequence of SEQ ID NO: 820 and an antisense strandcomprising the sequence of SEQ ID NO: 2323; (f) a sense strandcomprising the sequence of SEQ ID NO: 1147 and an antisense strandcomprising the sequence of SEQ ID NO: 2650; (g) a sense strandcomprising the sequence of SEQ ID NO: 1148 and an antisense strandcomprising the sequence of SEQ ID NO: 2651; (h) a sense strandcomprising the sequence of SEQ ID NO: 1364 and an antisense strandcomprising the sequence of SEQ ID NO: 2867; (i) a sense strandcomprising the sequence of SEQ ID NO: 1366 and an antisense strandcomprising the sequence of SEQ ID NO: 2869; or (j) a sense strandcomprising the sequence of SEQ ID NO: 1370 and an antisense strandcomprising the sequence of SEQ ID NO: 2873.

The RNAi construct of the invention can be any of the duplex compoundslisted in Tables 1 to 10 (including the nucleotide sequences and/orchemical modifications of the compounds). In some embodiments, the RNAiconstruct is any of the duplex compounds listed in Table 1. In otherembodiments, the RNAi construct is any of the duplex compounds listed inTable 6 (including the nucleotide sequences and/or chemicalmodifications of the compounds). In still other embodiments, the RNAiconstruct is any of the duplex compounds listed in Table 8 (includingthe nucleotide sequences and/or chemical modifications of thecompounds). In certain embodiments, the RNAi construct is D-1098,D-1176, D-1200, D-1206, D-1235, D-1246, D-1373, D-1389, D-1813, D-1815,D-1983, D-2000, D-2045, D-2142, D-2143, D-1438, D-1494, D-2357, D-2359,D-2361, D-2365, D-2461, D-3036, D-3037, D-3051, D-3053, D-3057, D-3779,D-3780, D-3782, D-3788, D-3791, D-3795, D-3799, or D-3800. In someembodiments, the RNAi construct is D-1200, D-1206, D-1235, D-1815,D-2143, D-2359, D-2361, D-2365, D-2142, D-1176, D-3779, D-3782, D-3788,D-3799, or D-3800. In one particular embodiment, the RNAi construct isD-1235. In another particular embodiment, the RNAi construct is D-2143.In another embodiment, the RNAi construct is D-2361. In anotherembodiment, the RNAi construct is D-1815. In another embodiment, theRNAi construct is D-2359. In still another embodiment, the RNAiconstruct is D-3782. In yet another embodiment, the RNAi construct isD-3799.

In certain embodiments, the antisense strands of the RNAi constructs ofthe invention may target certain regions of the ASGR1 mRNA sequence. Forinstance, in some embodiments, the antisense strand of an RNAi constructof the invention comprises a sequence that is substantiallycomplementary or fully complementary to nucleotides 692 to 721 of thehuman ASGR1 mRNA transcript set forth in SEQ ID NO: 1, nucleotides 692to 716 of the human ASGR1 mRNA transcript set forth in SEQ ID NO: 1, ornucleotides 692 to 710 of the human ASGR1 mRNA transcript set forth inSEQ ID NO: 1. In such embodiments, the RNAi construct may comprise asense strand that is substantially complementary or fully complementaryto the antisense strand targeting this region. Thus, in theseembodiments, the sense strand may comprise a sequence identical tonucleotides 692 to 721, nucleotides 692 to 716, or nucleotides 692 to710 of SEQ ID NO: 1.

In other embodiments, the antisense strand of an RNAi construct of theinvention comprises a sequence that is substantially complementary orfully complementary to nucleotides 396 to 425 of the human ASGR1 mRNAtranscript set forth in SEQ ID NO: 1, nucleotides 396 to 420 of thehuman ASGR1 mRNA transcript set forth in SEQ ID NO: 1, or nucleotides396 to 414 of the human ASGR1 mRNA transcript set forth in SEQ ID NO: 1.In such embodiments, the RNAi construct may comprise a sense strand thatis substantially complementary or fully complementary to the antisensestrand targeting this region. Thus, in these embodiments, the sensestrand may comprise a sequence identical to nucleotides 396 to 425,nucleotides 396 to 420, or nucleotides 396 to 414 of SEQ ID NO: 1.

In still other embodiments, the antisense strand of an RNAi construct ofthe invention comprises a sequence that is substantially complementaryor fully complementary to nucleotides 886 to 915 of the human ASGR1 mRNAtranscript set forth in SEQ ID NO: 1, nucleotides 886 to 910 of thehuman ASGR1 mRNA transcript set forth in SEQ ID NO: 1, or nucleotides886 to 904 of the human ASGR1 mRNA transcript set forth in SEQ ID NO: 1.In such embodiments, the RNAi construct may comprise a sense strand thatis substantially complementary or fully complementary to the antisensestrand targeting this region. Thus, in these embodiments, the sensestrand may comprise a sequence identical to nucleotides 886 to 915,nucleotides 886 to 910, or nucleotides 886 to 904 of SEQ ID NO: 1.

The RNAi constructs of the invention may comprise one or more modifiednucleotides. A “modified nucleotide” refers to a nucleotide that has oneor more chemical modifications to the nucleoside, nucleobase, pentosering, or phosphate group. As used herein, modified nucleotides do notencompass ribonucleotides containing adenosine monophosphate, guanosinemonophosphate, uridine monophosphate, and cytidine monophosphate, anddeoxyribonucleotides containing deoxyadenosine monophosphate,deoxyguanosine monophosphate, deoxythymidine monophosphate, anddeoxycytidine monophosphate.

However, the RNAi constructs may comprise combinations of modifiednucleotides, ribonucleotides, and deoxyribonucleotides. Incorporation ofmodified nucleotides into one or both strands of double-stranded RNAmolecules can improve the in vivo stability of the RNA molecules, e.g.,by reducing the molecules' susceptibility to nucleases and otherdegradation processes. The potency of RNAi constructs for reducingexpression of the target gene can also be enhanced by incorporation ofmodified nucleotides.

In certain embodiments, the modified nucleotides have a modification ofthe ribose sugar. These sugar modifications can include modifications atthe 2′ and/or 5′ position of the pentose ring as well as bicyclic sugarmodifications. A 2′-modified nucleotide refers to a nucleotide having apentose ring with a substituent at the 2′ position other than H or OH.Such 2′-modifications include, but are not limited to, 2′-O-alkyl (e.g.O—C₁-C₁₀ or O—C₁-C₁₀ substituted alkyl), 2′-O-allyl (O—CH₂CH═CH₂),2′-C-allyl, 2′-fluoro, 2′-O-methyl (OCH₃), 2′-O-methoxyethyl(O—(CH₂)₂OCH₃), 2′-OCF₃, 2′-O(CH₂)₂SCH₃, 2′-O-aminoalkyl, 2′-amino (e.g.NH₂), 2′-O-ethylamine, and 2′-azido. Modifications at the 5′ position ofthe pentose ring include, but are not limited to, 5′-methyl (R or S);5′-vinyl, and 5′-methoxy.

A “bicyclic sugar modification” refers to a modification of the pentosering where a bridge connects two atoms of the ring to form a second ringresulting in a bicyclic sugar structure. In some embodiments thebicyclic sugar modification comprises a bridge between the 4′ and 2′carbons of the pentose ring. Nucleotides comprising a sugar moiety witha bicyclic sugar modification are referred to herein as bicyclic nucleicacids or BNAs. Exemplary bicyclic sugar modifications include, but arenot limited to, α-L-Methyleneoxy (4′-CH₂—O-2′) bicyclic nucleic acid(BNA); β-D-Methyleneoxy (4′-CH₂—O-2′) BNA (also referred to as a lockednucleic acid or LNA); Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA; Aminooxy(4′-CH₂—O—N(R)-2′) BNA; Oxyamino (4′-CH₂—N(R)—O-2′) BNA;Methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to asconstrained ethyl or cEt); methylene-thio (4′-CH₂—S-2′) BNA;methylene-amino (4′-CH₂—N(R)-2′) BNA; methyl carbocyclic(4′-CH₂—CH(CH₃)-2′) BNA; propylene carbocyclic (4′-(CH₂)₃-2′) BNA; andMethoxy(ethyleneoxy) (4′-CH(CH₂OMe)-O-2′) BNA (also referred to asconstrained MOE or cMOE). These and other sugar-modified nucleotidesthat can be incorporated into the RNAi constructs of the invention aredescribed in U.S. Pat. No. 9,181,551, U.S. Patent Publication No.2016/0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are hereby incorporated by reference intheir entireties.

In some embodiments, the RNAi constructs comprise one or more 2′-fluoromodified nucleotides, 2′-O-methyl modified nucleotides,2′-O-methoxyethyl modified nucleotides, 2′-O-allyl modified nucleotides,bicyclic nucleic acids (BNAs), or combinations thereof. In certainembodiments, the RNAi constructs comprise one or more 2′-fluoro modifiednucleotides, 2′-O-methyl modified nucleotides, 2′-O-methoxyethylmodified nucleotides, or combinations thereof. In one particularembodiment, the RNAi constructs comprise one or more 2′-fluoro modifiednucleotides, 2′-O-methyl modified nucleotides or combinations thereof.

Both the sense and antisense strands of the RNAi constructs can compriseone or multiple modified nucleotides. For instance, in some embodiments,the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moremodified nucleotides. In certain embodiments, all nucleotides in thesense strand are modified nucleotides. In some embodiments, theantisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moremodified nucleotides. In other embodiments, all nucleotides in theantisense strand are modified nucleotides. In certain other embodiments,all nucleotides in the sense strand and all nucleotides in the antisensestrand are modified nucleotides. In these and other embodiments, themodified nucleotides can be 2′-fluoro modified nucleotides, 2′-O-methylmodified nucleotides, or combinations thereof.

In some embodiments, all pyrimidine nucleotides preceding an adenosinenucleotide in the sense strand, antisense strand, or both strands aremodified nucleotides. For example, where the sequence 5′-CA-3′ or5′-UA-3′ appears in either strand, the cytidine and uridine nucleotidesare modified nucleotides, preferably 2′-O-methyl modified nucleotides.In certain embodiments, all pyrimidine nucleotides in the sense strandare modified nucleotides (e.g. 2′-O-methyl modified nucleotides), andthe 5′ nucleotide in all occurrences of the sequence 5′-CA-3′ or5′-UA-3′ in the antisense strand are modified nucleotides (e.g.2′-O-methyl modified nucleotides). In other embodiments, all nucleotidesin the duplex region are modified nucleotides. In such embodiments, themodified nucleotides are preferably 2′-O-methyl modified nucleotides,2′-fluoro modified nucleotides or combinations thereof.

In embodiments in which the RNAi construct comprises a nucleotideoverhang, the nucleotides in the overhang can be ribonucleotides,deoxyribonucleotides, or modified nucleotides. In one embodiment, thenucleotides in the overhang are deoxyribonucleotides, e.g.deoxythymidine. In another embodiment, the nucleotides in the overhangare modified nucleotides. For instance, in some embodiments, thenucleotides in the overhang are 2′-O-methyl modified nucleotides,2′-fluoro modified nucleotides, 2′-methoxyethyl modified nucleotides, orcombinations thereof.

The RNAi constructs of the invention may also comprise one or moremodified internucleotide linkages. As used herein, the term “modifiedinternucleotide linkage” refers to an internucleotide linkage other thanthe natural 3′ to 5′ phosphodiester linkage. In some embodiments, themodified internucleotide linkage is a phosphorous-containinginternucleotide linkage, such as a phosphotriester,aminoalkylphosphotriester, an alkylphosphonate (e.g. methylphosphonate,3′-alkylene phosphonate), a phosphinate, a phosphoramidate (e.g.3′-amino phosphoramidate and aminoalkylphosphoramidate), aphosphorothioate (P═S), a chiral phosphorothioate, a phosphorodithioate,a thionophosphoramidate, a thionoalkylphosphonate, athionoalkylphosphotriester, and a boranophosphate. In one embodiment, amodified internucleotide linkage is a 2′ to 5′ phosphodiester linkage.In other embodiments, the modified internucleotide linkage is anon-phosphorous-containing internucleotide linkage and thus can bereferred to as a modified internucleoside linkage. Suchnon-phosphorous-containing linkages include, but are not limited to,morpholino linkages (formed in part from the sugar portion of anucleoside); siloxane linkages (—O—Si(H)₂—O—); sulfide, sulfoxide andsulfone linkages; formacetyl and thioformacetyl linkages; alkenecontaining backbones; sulfamate backbones; methylenemethylimino(—CH₂—N(CH₃)—O—CH₂—) and methylenehydrazino linkages; sulfonate andsulfonamide linkages; amide linkages; and others having mixed N, O, Sand CH₂ component parts. In one embodiment, the modified internucleosidelinkage is a peptide-based linkage (e.g. aminoethylglycine) to create apeptide nucleic acid or PNA, such as those described in U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262. Other suitable modifiedinternucleotide and internucleoside linkages that may be employed in theRNAi constructs of the invention are described in U.S. Pat. Nos.6,693,187, 9,181,551, U.S. Patent Publication No. 2016/0122761, andDeleavey and Damha, Chemistry and Biology, Vol. 19: 937-954, 2012, allof which are hereby incorporated by reference in their entireties.

In certain embodiments, the RNAi constructs comprise one or morephosphorothioate internucleotide linkages. The phosphorothioateinternucleotide linkages may be present in the sense strand, antisensestrand, or both strands of the RNAi constructs. For instance, in someembodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or morephosphorothioate internucleotide linkages. In other embodiments, theantisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or morephosphorothioate internucleotide linkages. In still other embodiments,both strands comprise 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioateinternucleotide linkages. The RNAi constructs can comprise one or morephosphorothioate internucleotide linkages at the 3′-end, the 5′-end, orboth the 3′- and 5′-ends of the sense strand, the antisense strand, orboth strands. For instance, in certain embodiments, the RNAi constructcomprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 ormore) consecutive phosphorothioate internucleotide linkages at the3′-end of the sense strand, the antisense strand, or both strands. Inother embodiments, the RNAi construct comprises about 1 to about 6 ormore (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioateinternucleotide linkages at the 5′-end of the sense strand, theantisense strand, or both strands. In one embodiment, the RNAi constructcomprises a single phosphorothioate internucleotide linkage at the 3′end of the sense strand and a single phosphorothioate internucleotidelinkage at the 3′ end of the antisense strand. In another embodiment,the RNAi construct comprises two consecutive phosphorothioateinternucleotide linkages at the 3′ end of the antisense strand (i.e. aphosphorothioate internucleotide linkage at the first and secondinternucleotide linkages at the 3′ end of the antisense strand). Inanother embodiment, the RNAi construct comprises two consecutivephosphorothioate internucleotide linkages at both the 3′ and 5′ ends ofthe antisense strand. In yet another embodiment, the RNAi constructcomprises two consecutive phosphorothioate internucleotide linkages atboth the 3′ and 5′ ends of the antisense strand and two consecutivephosphorothioate internucleotide linkages at the 5′ end of the sensestrand. In still another embodiment, the RNAi construct comprises twoconsecutive phosphorothioate internucleotide linkages at both the 3′ and5′ ends of the antisense strand and two consecutive phosphorothioateinternucleotide linkages at both the 3′ and 5′ ends of the sense strand(i.e. a phosphorothioate internucleotide linkage at the first and secondinternucleotide linkages at both the 5′ and 3′ ends of the antisensestrand and a phosphorothioate internucleotide linkage at the first andsecond internucleotide linkages at both the 5′ and 3′ ends of the sensestrand). In any of the embodiments in which one or both strandscomprises one or more phosphorothioate internucleotide linkages, theremaining internucleotide linkages within the strands can be the natural3′ to 5′ phosphodiester linkages. For instance, in some embodiments,each internucleotide linkage of the sense and antisense strands isselected from phosphodiester and phosphorothioate, wherein at least oneinternucleotide linkage is a phosphorothioate.

In embodiments in which the RNAi construct comprises a nucleotideoverhang, two or more of the unpaired nucleotides in the overhang can beconnected by a phosphorothioate internucleotide linkage. In certainembodiments, all the unpaired nucleotides in a nucleotide overhang atthe 3′ end of the anti sense strand and/or the sense strand areconnected by phosphorothioate internucleotide linkages. In otherembodiments, all the unpaired nucleotides in a nucleotide overhang atthe 5′ end of the antisense strand and/or the sense strand are connectedby phosphorothioate internucleotide linkages. In still otherembodiments, all the unpaired nucleotides in any nucleotide overhang areconnected by phosphorothioate internucleotide linkages.

In certain embodiments, the modified nucleotides incorporated into oneor both of the strands of the RNAi constructs of the invention have amodification of the nucleobase (also referred to herein as “base”). A“modified nucleobase” or “modified base” refers to a base other than thenaturally occurring purine bases adenine (A) and guanine (G) andpyrimidine bases thymine (T), cytosine (C), and uracil (U). Modifiednucleobases can be synthetic or naturally occurring modifications andinclude, but are not limited to, universal bases, 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine (X), hypoxanthine (I),2-aminoadenine, 6-methyladenine, 6-methylguanine, and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine.

In some embodiments, the modified base is a universal base. A “universalbase” refers to a base analog that indiscriminately forms base pairswith all of the natural bases in RNA and DNA without altering the doublehelical structure of the resulting duplex region. Universal bases areknown to those of skill in the art and include, but are not limited to,inosine, C-phenyl, C-naphthyl and other aromatic derivatives, azolecarboxamides, and nitroazole derivatives, such as 3-nitropyrrole,4-nitroindole, 5-nitroindole, and 6-nitroindole.

Other suitable modified bases that can be incorporated into the RNAiconstructs of the invention include those described in Herdewijn,Antisense Nucleic Acid Drug Dev., Vol. 10: 297-310, 2000 and Peacock etal., J. Org. Chem., Vol. 76: 7295-7300, 2011, both of which are herebyincorporated by reference in their entireties. The skilled person iswell aware that guanine, cytosine, adenine, thymine, and uracil may bereplaced by other nucleobases, such as the modified nucleobasesdescribed above, without substantially altering the base pairingproperties of a polynucleotide comprising a nucleotide bearing suchreplacement nucleobase.

In some embodiments, the sense and antisense strands of the RNAiconstructs may comprise one or more abasic nulceotides. An “abasicnucleotide” or “abasic nucleoside” is a nucleotide or nucleoside thatlacks a nucleobase at the 1′ position of the ribose sugar. In certainembodiments, the abasic nucleotides are incorporated into the terminalends of the sense and/or antisense strands of the RNAi constructs. Inone embodiment, the sense strand comprises an abasic nucleotide as theterminal nucleotide at its 3′ end, its 5′ end, or both its 3′ and 5′ends. In another embodiment, the antisense strand comprises an abasicnucleotide as the terminal nucleotide at its 3′ end, its 5′ end, or bothits 3′ and 5′ ends. In such embodiments in which the abasic nucleotideis a terminal nucleotide, it may be linked to the adjacent nucleotidethrough a 3′-3′ internucleotide linkage (i.e. an inverted nucleotide)rather than the natural 3′-5′ internucleotide linkage.

In some embodiments of the RNAi constructs of the invention, the 5′ endof the sense strand, antisense strand, or both the antisense and sensestrands comprises a phosphate moiety. As used herein, the term“phosphate moiety” refers to a terminal phosphate group that includesunmodified phosphates (—O—P═O)(OH)OH) as well as modified phosphates.Modified phosphates include phosphates in which one or more of the O andOH groups is replaced with H, O, S, N(R) or alkyl where R is H, an aminoprotecting group or unsubstituted or substituted alkyl. Exemplaryphosphate moieties include, but are not limited to, 5′-monophosphate;5′-diphosphate; 5′-triphosphate; 5′-guanosine cap (7-methylated ornon-methylated); 5′-adenosine cap or any other modified or unmodifiednucleotide cap structure; 5′-monothiophosphate (phosphorothioate);5′-monodithiophosphate (phosphorodithioate); 5′-alpha-thiotriphosphate;5′-gamma-thiotriphosphate, 5′-phosphoramidates; 5′-vinylphosphates;5′-alkylphosphonates (e.g., alkyl=methyl, ethyl, isopropyl, propyl,etc.); and 5′-alkyletherphosphonates (e.g., alkylether=methoxymethyl,ethoxymethyl, etc.).

The modified nucleotides that can be incorporated into the RNAiconstructs of the invention may have more than one chemical modificationdescribed herein. For instance, the modified nucleotide may have amodification to the ribose sugar as well as a modification to thenucleobase. By way of example, a modified nucleotide may comprise a 2′sugar modification (e.g. 2′-fluoro or 2′-methyl) and comprise a modifiedbase (e.g. 5-methyl cytosine or pseudouracil). In other embodiments, themodified nucleotide may comprise a sugar modification in combinationwith a modification to the 5′ phosphate that would create a modifiedinternucleotide or internucleoside linkage when the modified nucleotidewas incorporated into a polynucleotide. For instance, in someembodiments, the modified nucleotide may comprise a sugar modification,such as a 2′-fluoro modification, a 2′-O-methyl modification, or abicyclic sugar modification, as well as a 5′ phosphorothioate group.Accordingly, in some embodiments, one or both strands of the RNAiconstructs of the invention comprise a combination of 2′ modifiednucleotides or BNAs and phosphorothioate internucleotide linkages. Incertain embodiments, both the sense and antisense strands of the RNAiconstructs of the invention comprise a combination of 2′-fluoro modifiednucleotides, 2′-O-methyl modified nucleotides, and phosphorothioateinternucleotide linkages. Exemplary RNAi constructs comprising modifiednucleotides and internucleotide linkages are shown in Tables 6 and 8.

Preferably, the RNAi constructs of the invention reduce or inhibit theexpression of ASGR1 in cells, particularly liver cells. Accordingly, inone embodiment, the present invention provides a method of reducingASGR1 expression in a cell by contacting the cell with any RNAiconstruct described herein. The cell may be in vitro or in vivo. ASGR1expression can be assessed by measuring the amount or level of ASGR1mRNA, ASGR1 protein, or another biomarker linked to ASGR1 expression,such as serum levels of alkaline phosphatase. The reduction of ASGR1expression in cells or animals treated with an RNAi construct of theinvention can be determined relative to the ASGR1 expression in cells oranimals not treated with the RNAi construct or treated with a controlRNAi construct. For instance, in some embodiments, reduction of ASGR1expression is assessed by (a) measuring the amount or level of ASGR1mRNA in liver cells treated with a RNAi construct of the invention, (b)measuring the amount or level of ASGR1 mRNA in liver cells treated witha control RNAi construct (e.g. RNAi agent directed to a RNA molecule notexpressed in liver cells or a RNAi construct having a nonsense orscrambled sequence) or no construct, and (c) comparing the measuredASGR1 mRNA levels from treated cells in (a) to the measured ASGR1 mRNAlevels from control cells in (b). The ASGR1 mRNA levels in the treatedcells and controls cells can be normalized to RNA levels for a controlgene (e.g. 18S ribosomal RNA or housekeeping gene) prior to comparison.ASGR1 mRNA levels can be measured by a variety of methods, includingNorthern blot analysis, nuclease protection assays, fluorescence in situhybridization (FISH), reverse-transcriptase (RT)-PCR, real-time RT-PCR,quantitative PCR, droplet digital PCR, and the like.

In other embodiments, reduction of ASGR1 expression is assessed by (a)measuring the amount or level of ASGR1 protein in liver cells treatedwith a RNAi construct of the invention, (b) measuring the amount orlevel of ASGR1 protein in liver cells treated with a control RNAiconstruct (e.g. RNAi agent directed to a RNA molecule not expressed inliver cells or a RNAi construct having a nonsense or scrambled sequence)or no construct, and (c) comparing the measured ASGR1 protein levelsfrom treated cells in (a) to the measured ASGR1 protein levels fromcontrol cells in (b). Methods of measuring ASGR1 protein levels areknown to those of skill in the art, and include Western Blots,immunoassays (e.g. ELISA), and flow cytometry. An exemplaryimmunoassay-based method for assessing ASGR1 protein expression isdescribed in Examples 2 and 7. Example 3 describes an exemplary methodfor measuring ASGR1 mRNA using RNA FISH, and Example 8 describes anexemplary method for assessing ASGR1 mRNA using droplet digital PCR. Anymethod capable of measuring ASGR1 mRNA or protein can be used to assessthe efficacy of the RNAi constructs of the invention.

In some embodiments, the methods to assess ASGR1 expression levels areperformed in vitro in cells that natively express ASGR1 (e.g. livercells) or cells that have been engineered to express ASGR1. In certainembodiments, the methods are performed in vitro in liver cells. Suitableliver cells include, but are not limited to, primary hepatocytes (e.g.human, non-human primate, or rodent hepatocytes), HepAD38 cells, HuH-6cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2cells. In one embodiment, the liver cells are Hep3B cells. In anotherembodiment, the liver cells are human primary hepatopyctes.

In other embodiments, the methods to assess ASGR1 expression levels areperformed in vivo. The RNAi constructs and any control RNAi constructscan be administered to an animal (e.g. rodent or non-human primate) andASGR1 mRNA or protein levels assessed in liver tissue harvested from theanimal following treatment. Alternatively or additionally, a biomarkeror functional phenotype associated with ASGR1 expression can be assessedin the treated animals. For instance, elevated serum alkalinephosphatase levels correlate with reduced serum levels of non-HDLcholesterol in individuals with loss of function mutations in the ASGR1gene (Nioi et al., New England Journal of Medicine, Vol.374(22):2131-2141, 2016, which is hereby incorporated by reference inits entirety). Thus, serum levels of alkaline phosphatase or non-HDLcholesterol can be measured in animals treated with RNAi constructs ofthe invention to assess the functional efficacy of reducing ASGR1expression. Exemplary methods for these analyses are described inExamples 6, 9, and 10.

In certain embodiments, expression of ASGR1 is reduced in liver cells byat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, or at least 50% by an RNAiconstruct of the invention. In some embodiments, expression of ASGR1 isreduced in liver cells by at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, or at least 85% by an RNAi construct of theinvention. In other embodiments, the expression of ASGR1 is reduced inliver cells by about 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more by an RNAi construct of the invention. Thepercent reduction of ASGR1 expression can be measured by any of themethods described herein as well as others known in the art. Forinstance, in certain embodiments, the RNAi constructs of the inventioninhibit at least 45% of ASGR1 expression at 5 nM in Hep3B cells invitro. In related embodiments, the RNAi constructs of the inventioninhibit at least 50%, at least 55%, at least 60%, at least 65%, at least70%, or at least 75% of ASGR1 expression at 5 nM in Hep3B cells invitro. In other embodiments, the RNAi constructs of the inventioninhibit at least 80%, at least 85%, at least 90%, at least 92%, at least94%, at least 96%, or at least 98% of ASGR1 expression at 5 nM in Hep3Bcells in vitro.

In some embodiments, an IC50 value is calculated to assess the potencyof an RNAi construct of the invention for inhibiting ASGR1 expression inliver cells. An “IC50 value” is the dose/concentration required toachieve 50% inhibition of a biological or biochemical function. The IC50value of any particular substance or antagonist can be determined byconstructing a dose-response curve and examining the effect of differentconcentrations of the substance or antagonist on expression levels orfunctional activity in any assay. IC50 values can be calculated for agiven antagonist or substance by determining the concentration needed toinhibit half of the maximum biological response or native expressionlevels. Thus, the IC50 value for any RNAi construct can be calculated bydetermining the concentration of the RNAi construct needed to inhibithalf of the native ASGR1 expression level in liver cells (e.g. ASGR1expression level in control liver cells) in any assay, such as theimmunoassay, RNA FISH assay, qPCR or droplet digital PCR assaysdescribed in the Examples. The RNAi constructs of the invention mayinhibit ASGR1 expression in liver cells (e.g. Hep3B cells) with an IC50of less than about 10 nM, less than about 5 nM, or less than about 1 nM.For example, the RNAi constructs inhibit ASGR1 expression in liver cellswith an IC50 of about 0.5 nM to about 10 nM, about 0.8 nM to about 8 nM,about 1 nM to about 5 nM, about 0.8 nM to about 3 nM, about 0.001 nM toabout 1 nM, about 0.001 nM to about 0.50 nM, about 0.001 nM to about 0.1nM, about 0.001 nM to about 0.01 nM, about 0.01 nM to about 0.50 nM,about 0.02 nM to about 0.80 nM, about 0.01 nM to about 1.0 nM, about 0.1nM to about 0.9 nM, or about 0.05 nM to about 0.5 nM. In certainembodiments, the RNAi construct inhibits ASGR1 expression in liver cells(e.g. Hep3B cells) with an IC50 of about 0.5 nM to about 5 nM. In otherembodiments, the RNAi construct inhibits ASGR1 expression in liver cells(e.g. Hep3B cells) with an IC50 of about 0.01 nM to about 0.9 nM.

The RNAi constructs of the invention can readily be made usingtechniques known in the art, for example, using conventional nucleicacid solid phase synthesis. The polynucleotides of the RNAi constructscan be assembled on a suitable nucleic acid synthesizer utilizingstandard nucleotide or nucleoside precursors (e.g. phosphoramidites).Automated nucleic acid synthesizers are sold commercially by severalvendors, including DNA/RNA synthesizers from Applied Biosystems (FosterCity, Calif.), MerMade synthesizers from BioAutomation (Irving, Tex.),and OligoPilot synthesizers from GE Healthcare Life Sciences(Pittsburgh, Pa.).

The 2′ silyl protecting group can be used in conjunction with acidlabile dimethoxytrityl (DMT) at the 5′ position of ribonucleosides tosynthesize oligonucleotides via phosphoramidite chemistry. Finaldeprotection conditions are known not to significantly degrade RNAproducts. All syntheses can be conducted in any automated or manualsynthesizer on large, medium, or small scale. The syntheses may also becarried out in multiple well plates, columns, or glass slides.

The 2′-O-silyl group can be removed via exposure to fluoride ions, whichcan include any source of fluoride ion, e.g., those salts containingfluoride ion paired with inorganic counterions e.g., cesium fluoride andpotassium fluoride or those salts containing fluoride ion paired with anorganic counterion, e.g., a tetraalkylammonium fluoride. A crown ethercatalyst can be utilized in combination with the inorganic fluoride inthe deprotection reaction. Preferred fluoride ion source aretetrabutylammonium fluoride or aminohydrofluorides (e.g., combiningaqueous HF with triethylamine in a dipolar aprotic solvent, e.g.,dimethylformamide).

The choice of protecting groups for use on the phosphite triesters andphosphotriesters can alter the stability of the triesters towardsfluoride. Methyl protection of the phosphotriester or phosphitetriestercan stabilize the linkage against fluoride ions and improve processyields.

Since ribonucleosides have a reactive 2′ hydroxyl substituent, it can bedesirable to protect the reactive 2′ position in RNA with a protectinggroup that is orthogonal to a 5′-O-dimethoxytrityl protecting group,e.g., one stable to treatment with acid. Silyl protecting groups meetthis criterion and can be readily removed in a final fluoridedeprotection step that can result in minimal RNA degradation.

Tetrazole catalysts can be used in the standard phosphoramidite couplingreaction. Preferred catalysts include, e.g., tetrazole,S-ethyl-tetrazole, benzylthiotetrazole, p-nitrophenyltetrazole.

As can be appreciated by the skilled artisan, further methods ofsynthesizing the RNAi constructs described herein will be evident tothose of ordinary skill in the art. Additionally, the various syntheticsteps may be performed in an alternate sequence or order to give thedesired compounds. Other synthetic chemistry transformations, protectinggroups (e.g., for hydroxyl, amino, etc. present on the bases) andprotecting group methodologies (protection and deprotection) useful insynthesizing the RNAi constructs described herein are known in the artand include, for example, those such as described in R. Larock,Comprehensive Organic Transformations, VCH Publishers (1989); T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and subsequent editions thereof. Custom synthesisof RNAi agents is also available from several commercial vendors,including Dharmacon, Inc. (Lafayette, Colo.), AxoLabs GmbH (Kulmbach,Germany), and Ambion, Inc. (Foster City, Calif.).

The RNAi constructs of the invention may comprise a ligand. As usedherein, a “ligand” refers to any compound or molecule that is capable ofinteracting with another compound or molecule, directly or indirectly.The interaction of a ligand with another compound or molecule may elicita biological response (e.g. initiate a signal transduction cascade,induce receptor-mediated endocytosis) or may just be a physicalassociation. The ligand can modify one or more properties of thedouble-stranded RNA molecule to which is attached, such as thepharmacodynamic, pharmacokinetic, binding, absorption, cellulardistribution, cellular uptake, charge and/or clearance properties of theRNA molecule.

The ligand may comprise a serum protein (e.g., human serum albumin,low-density lipoprotein, globulin), a cholesterol moiety, a vitamin(biotin, vitamin E, vitamin B₁₂), a folate moiety, a steroid, a bileacid (e.g. cholic acid), a fatty acid (e.g., palmitic acid, myristicacid), a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid), a glycoside, a phospholipid,or antibody or binding fragment thereof (e.g. antibody or bindingfragment that targets the RNAi construct to a specific cell type, suchas liver). Other examples of ligands include dyes, intercalating agents(e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipophilic molecules, e.g, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl,or phenoxazine), peptides (e.g., antennapedia peptide, Tat peptide, RGDpeptides), alkylating agents, polymers, such as polyethylene glycol(PEG)(e.g., PEG-40K), polyamino acids, and polyamines (e.g. spermine,spermidine).

In certain embodiments, the ligands have endosomolytic properties. Theendosomolytic ligands promote the lysis of the endosome and/or transportof the RNAi construct of the invention, or its components, from theendosome to the cytoplasm of the cell. The endosomolytic ligand may be apolycationic peptide or peptidomimetic, which shows pH-dependentmembrane activity and fusogenicity. In one embodiment, the endosomolyticligand assumes its active conformation at endosomal pH. The “active”conformation is that conformation in which the endosomolytic ligandpromotes lysis of the endosome and/or transport of the RNAi construct ofthe invention, or its components, from the endosome to the cytoplasm ofthe cell. Exemplary endosomolytic ligands include the GALA peptide(Subbarao et al., Biochemistry, Vol. 26: 2964-2972, 1987), the EALApeptide (Vogel et al., J. Am. Chem. Soc., Vol. 118: 1581-1586, 1996),and their derivatives (Turk et al., Biochem. Biophys. Acta, Vol. 1559:56-68, 2002). In one embodiment, the endosomolytic component may containa chemical group (e.g., an amino acid) which will undergo a change incharge or protonation in response to a change in pH. The endosomolyticcomponent may be linear or branched.

In some embodiments, the ligand comprises a lipid or other hydrophobicmolecule. In one embodiment, the ligand comprises a cholesterol moietyor other steroid. Cholesterol-conjugated oligonucleotides have beenreported to be more active than their unconjugated counterparts(Manoharan, Antisense Nucleic Acid Drug Development, Vol. 12: 103-228,2002). Ligands comprising cholesterol moieties and other lipids forconjugation to nucleic acid molecules have also been described in U.S.Pat. Nos. 7,851,615; 7,745,608; and 7,833,992, all of which are herebyincorporated by reference in their entireties. In another embodiment,the ligand comprises a folate moiety. Polynucleotides conjugated tofolate moieties can be taken up by cells via a receptor-mediatedendocytosis pathway. Such folate-polynucleotide conjugates are describedin U.S. Pat. No. 8,188,247, which is hereby incorporated by reference inits entirety.

Given that ASGR1 is expressed on the surface of liver cells (e.g.hepatocytes) as a component of the asialoglycoprotein receptor (ASGR),in certain embodiments, it is desirable to specifically deliver the RNAiconstruct to those liver cells. Accordingly, in certain embodiments, theligand targets delivery of the RNAi constructs specifically to livercells (e.g. hepatocytes) using various approaches as described in moredetail below. In certain embodiments, the RNAi constructs are targetedto liver cells with a ligand that binds to the surface-expressed ASGR,ASGR1 and/or ASGR2. In these embodiments, it is envisioned that thistargeting approach can result in a self-regulating system that reducesthe amount of RNAi construct delivered to the liver cells as expressionof ASGR1 is reduced due to the effect of the previously delivered RNAiconstruct.

In some embodiments, RNAi constructs can be specifically targeted to theliver by employing ligands that bind to or interact with proteinsexpressed on the surface of liver cells. For example, in certainembodiments, the ligands may comprise antigen binding proteins (e.g.antibodies or binding fragments thereof (e.g. Fab, scFv)) thatspecifically bind to a receptor expressed on hepatocytes, such as theasialoglycoprotein receptor and the LDL receptor. In one particularembodiment, the ligand comprises an antibody or binding fragment thereofthat specifically binds to ASGR1 and/or ASGR2. In another embodiment,the ligand comprises a Fab fragment of an antibody that specificallybinds to ASGR1 and/or ASGR2. A “Fab fragment” is comprised of oneimmunoglobulin light chain (i.e. light chain variable region (VL) andconstant region (CL)) and the CH1 region and variable region (VH) of oneimmunoglobulin heavy chain. In another embodiment, the ligand comprisesa single-chain variable antibody fragment (scFv fragment) of an antibodythat specifically binds to ASGR1 and/or ASGR2. An “scFv fragment”comprises the VH and VL regions of an antibody, wherein these regionsare present in a single polypeptide chain, and optionally comprising apeptide linker between the VH and VL regions that enables the Fv to formthe desired structure for antigen binding. Exemplary antibodies andbinding fragments thereof that specifically bind to ASGR1 that can beused as ligands for targeting the RNAi constructs of the invention tothe liver are described in U.S. Patent Application No. 62/234,546 andWIPO Publication No. WO 2017/058944, both of which are herebyincorporated by reference in their entireties. Other antibodies orbinding fragments thereof that specifically bind to ASGR1, LDL receptor,or other liver surface-expressed proteins suitable for use as ligands inthe RNAi constructs of the invention are commercially available.

In some embodiments, the ligand comprises a cys monoclonal antibody(mAb) or antigen-binding fragment thereof. A “cys mAb” is a monoclonalantibody or antigen-binding fragment thereof in which at least one aminoacid in the light chain or heavy chain has been substituted with acysteine amino acid or at least one cysteine amino acid has beeninserted into the primary sequence of the light chain or heavy chain.The free thiol group in the side chain of the cysteine amino acidprovides a conjugation site to which the sense strand of the RNAiconstructs of the invention can be covalently linked. The cysteinesubstitutions/additions can be at the amino-terminus or carboxy-terminusof the light chain or heavy chain of the antibody or antigen-bindingfragment. Alternatively or additionally, the cysteinesubstitutions/additions can be located at an internal site within thelight chain or heavy chain so long as the cysteine substitution/additiondoes not affect the binding affinity of the antibody or antigen-bindingfragment to its target antigen (e.g. ASGR1). Exemplary amino acidswithin the heavy and light chains of antibodies that may be substitutedwith cysteine residues are described in WIPO Publication Nos. WO2006/034488 and WO 2007/022070, both of which are hereby incorporated byreference in their entireties. In certain embodiments, the ligandcomprises a cys mAb or antigen-binding fragment thereof thatspecifically binds to human ASGR1. An exemplary anti-ASGR1 cys mAb isdescribed in Example 10. In one embodiment, the ligand comprises ananti-ASGR1 antibody having a heavy chain and a light chain, wherein theheavy chain comprises the sequence of SEQ ID NO: 4696 and the lightchain comprises the sequence of SEQ ID NO: 4697. Anti-ASGR1 cys mAbs orantigen-binding fragments thereof may be covalently attached to the 5′end or 3′ end of the sense strand of an RNAi construct of the invention,optionally through any of the linkers described herein. In someembodiments, the anti-ASGR1 antibody-RNA molecule conjugate comprisesone copy of the interfering RNA molecule (e.g. siRNA or shRNA)(i.e. anRNAi-to-antibody ratio of 1). In other embodiments, the anti-ASGR1antibody-RNA molecule conjugate comprises two copies of the interferingRNA molecule (e.g. siRNA or shRNA)(i.e. an RNAi-to-antibody ratio of 2).

In certain embodiments, the ligand comprises a carbohydrate. A“carbohydrate” refers to a compound made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom. Carbohydrates include, but are not limitedto, the sugars (e.g., monosaccharides, disaccharides, trisaccharides,tetrasaccharides, and oligosaccharides containing from about 4, 5, 6, 7,8, or 9 monosaccharide units), and polysaccharides, such as starches,glycogen, cellulose and polysaccharide gums. In some embodiments, thecarbohydrate incorporated into the ligand is a monosaccharide selectedfrom a pentose, hexose, or heptose and di- and tri-saccharides includingsuch monosaccharide units. In other embodiments, the carbohydrateincorporated into the ligand is an amino sugar, such as galactosamine,glucosamine, N-acetylgalactosamine, and N-acetylglucosamine.

In some embodiments, the ligand comprises a hexose or hexosamine. Thehexose may be selected from glucose, galactose, mannose, fucose, orfructose. The hexosamine may be selected from fructosamine,galactosamine, glucosamine, or mannosamine. In certain embodiments, theligand comprises glucose, galactose, galactosamine, or glucosamine. Inone embodiment, the ligand comprises glucose, glucosamine, orN-acetylglucosamine. In another embodiment, the ligand comprisesgalactose, galactosamine, or N-acetyl-galactosamine. In particularembodiments, the ligand comprises N-acetyl-galactosamine. Ligandscomprising glucose, galactose, and N-acetyl-galactosamine (GalNAc) areparticularly effective in targeting compounds to liver cells becausesuch ligands bind to the ASGR expressed on the surface of hepatocytes.See, e.g., D'Souza and Devarajan, J. Control Release, Vol. 203: 126-139,2015. Examples of GalNAc- or galactose-containing ligands that can beincorporated into the RNAi constructs of the invention are described inU.S. Pat. Nos. 7,491,805; 8,106,022; and 8,877,917; U.S. PatentPublication No. 20030130186; and WIPO Publication No. WO 2013166155, allof which are hereby incorporated by reference in their entireties.

In certain embodiments, the ligand comprises a multivalent carbohydratemoiety. As used herein, a “multivalent carbohydrate moiety” refers to amoiety comprising two or more carbohydrate units capable ofindependently binding or interacting with other molecules. For example,a multivalent carbohydrate moiety comprises two or more binding domainscomprised of carbohydrates that can bind to two or more differentmolecules or two or more different sites on the same molecule. Thevalency of the carbohydrate moiety denotes the number of individualbinding domains within the carbohydrate moiety. For instance, the terms“monovalent,” “bivalent,” “trivalent,” and “tetravalent” with referenceto the carbohydrate moiety refer to carbohydrate moieties with one, two,three, and four binding domains, respectively. The multivalentcarbohydrate moiety may comprise a multivalent lactose moiety, amultivalent galactose moiety, a multivalent glucose moiety, amultivalent N-acetyl-galactosamine moiety, a multivalentN-acetyl-glucosamine moiety, a multivalent mannose moiety, or amultivalent fucose moiety. In some embodiments, the ligand comprises amultivalent galactose moiety. In other embodiments, the ligand comprisesa multivalent N-acetyl-galactosamine moiety. In these and otherembodiments, the multivalent carbohydrate moiety is bivalent, trivalent,or tetravalent. In such embodiments, the multivalent carbohydrate moietycan be bi-antennary or tri-antennary. In one particular embodiment, themultivalent N-acetyl-galactosamine moiety is trivalent or tetravalent.In another particular embodiment, the multivalent galactose moiety istrivalent or tetravalent. Exemplary trivalent and tetravalentGalNAc-containing ligands for incorporation into the RNAi constructs ofthe invention are described in detail below.

The ligand can be attached or conjugated to the RNA molecule of the RNAiconstruct directly or indirectly. For instance, in some embodiments, theligand is covalently attached directly to the sense or antisense strandof the RNAi construct. In other embodiments, the ligand is covalentlyattached via a linker to the sense or antisense strand of the RNAiconstruct. The ligand can be attached to nucleobases, sugar moieties, orinternucleotide linkages of polynucleotides (e.g. sense strand orantisense strand) of the RNAi constructs of the invention. Conjugationor attachment to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In certainembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a ligand. Conjugation or attachment to pyrimidinenucleobases or derivatives thereof can also occur at any position. Insome embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobasecan be attached to a ligand. Conjugation or attachment to sugar moietiesof nucleotides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a ligand include the 2′, 3′, and 5′carbon atoms. The 1′ position can also be attached to a ligand, such asin an abasic residue. Internucleotide linkages can also support ligandattachments. For phosphorus-containing linkages (e.g., phosphodiester,phosphorothioate, phosphorodithiotate, phosphoroamidate, and the like),the ligand can be attached directly to the phosphorus atom or to an O,N, or S atom bound to the phosphorus atom. For amine- oramide-containing internucleoside linkages (e.g., PNA), the ligand can beattached to the nitrogen atom of the amine or amide or to an adjacentcarbon atom.

In certain embodiments, the ligand may be attached to the 3′ or 5′ endof either the sense or antisense strand. In certain embodiments, theligand is covalently attached to the 5′ end of the sense strand. Inother embodiments, the ligand is covalently attached to the 3′ end ofthe sense strand. For example, in some embodiments, the ligand isattached to the 3′-terminal nucleotide of the sense strand. In certainsuch embodiments, the ligand is attached at the 3′-position of the3′-terminal nucleotide of the sense strand. In alternative embodiments,the ligand is attached near the 3′ end of the sense strand, but beforeone or more terminal nucleotides (i.e. before 1, 2, 3, or 4 terminalnucleotides). In some embodiments, the ligand is attached at the2′-position of the sugar of the 3′-terminal nucleotide of the sensestrand.

In certain embodiments, the ligand is attached to the sense or antisensestrand via a linker. A “linker” is an atom or group of atoms thatcovalently joins a ligand to a polynucleotide component of the RNAiconstruct. The linker may be from about 1 to about 30 atoms in length,from about 2 to about 28 atoms in length, from about 3 to about 26 atomsin length, from about 4 to about 24 atoms in length, from about 6 toabout 20 atoms in length, from about 7 to about 20 atoms in length, fromabout 8 to about 20 atoms in length, from about 8 to about 18 atoms inlength, from about 10 to about 18 atoms in length, and from about 12 toabout 18 atoms in length. In some embodiments, the linker may comprise abifunctional linking moiety, which generally comprises an alkyl moietywith two functional groups. One of the functional groups is selected tobind to the compound of interest (e.g. sense or antisense strand of theRNAi construct) and the other is selected to bind essentially anyselected group, such as a ligand as described herein. In certainembodiments, the linker comprises a chain structure or an oligomer ofrepeating units, such as ethylene glycol or amino acid units. Examplesof functional groups that are typically employed in a bifunctionallinking moiety include, but are not limited to, electrophiles forreacting with nucleophilic groups and nucleophiles for reacting withelectrophilic groups. In some embodiments, bifunctional linking moietiesinclude amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g.,double or triple bonds), and the like.

Linkers that may be used to attach a ligand to the sense or antisensestrand in the RNAi constructs of the invention include, but are notlimited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid,substituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl orsubstituted or unsubstituted C₂-C₁₀ alkynyl. Preferred substituentgroups for such linkers include, but are not limited to, hydroxyl,amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy,halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, the linkers are cleavable. A cleavable linker isone which is sufficiently stable outside the cell, but which upon entryinto a target cell is cleaved to release the two parts the linker isholding together. In some embodiments, the cleavable linker is cleavedat least 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70times, 80 times, 90 times, or more, or at least 100 times faster in thetarget cell or under a first reference condition (which can, e.g., beselected to mimic or represent intracellular conditions) than in theblood of a subject, or under a second reference condition (which can,e.g., be selected to mimic or represent conditions found in the blood orserum).

Cleavable linkers are susceptible to cleavage agents, e.g., pH, redoxpotential or the presence of degradative molecules. Generally, cleavageagents are more prevalent or found at higher levels or activities insidecells than in serum or blood. Examples of such degradative agentsinclude: redox agents which are selected for particular substrates orwhich have no substrate specificity, including, e.g., oxidative orreductive enzymes or reductive agents such as mercaptans, present incells, that can degrade a redox cleavable linker by reduction;esterases; endosomes or agents that can create an acidic environment,e.g., those that result in a pH of five or lower; enzymes that canhydrolyze or degrade an acid cleavable linker by acting as a generalacid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linker may comprise a moiety that is susceptible to pH. ThepH of human serum is 7.4, while the average intracellular pH is slightlylower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, inthe range of 5.5-6.0, and lysosomes have an even more acidic pH ataround 5.0. Some linkers will have a cleavable group that is cleaved ata preferred pH, thereby releasing the RNA molecule from the ligandinside the cell, or into the desired compartment of the cell.

A linker can include a cleavable group that is cleavable by a particularenzyme. The type of cleavable group incorporated into a linker candepend on the cell to be targeted. For example, liver-targeting ligandscan be linked to RNA molecules through a linker that includes an estergroup. Liver cells are rich in esterases, and therefore the linker willbe cleaved more efficiently in liver cells than in cell types that arenot esterase-rich. Other types of cells rich in esterases include cellsof the lung, renal cortex, and testis. Linkers that contain peptidebonds can be used when targeting cells rich in peptidases, such as livercells and synoviocytes.

In general, the suitability of a candidate cleavable linker can beevaluated by testing the ability of a degradative agent (or condition)to cleave the candidate linker. It will also be desirable to also testthe candidate cleavable linker for the ability to resist cleavage in theblood or when in contact with other non-target tissue. Thus, one candetermine the relative susceptibility to cleavage between a first and asecond condition, where the first is selected to be indicative ofcleavage in a target cell and the second is selected to be indicative ofcleavage in other tissues or biological fluids, e.g., blood or serum.The evaluations can be carried out in cell free systems, in cells, incell culture, in organ or tissue culture, or in whole animals. It may beuseful to make initial evaluations in cell-free or culture conditionsand to confirm by further evaluations in whole animals. In someembodiments, useful candidate linkers are cleaved at least 2, 4, 10, 20,50, 70, or 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

In other embodiments, redox cleavable linkers are utilized. Redoxcleavable linkers are cleaved upon reduction or oxidation. An example ofreductively cleavable group is a disulfide linking group (—S—S—). Todetermine if a candidate cleavable linker is a suitable “reductivelycleavable linker,” or for example is suitable for use with a particularRNAi construct and particular ligand, one can use one or more methodsdescribed herein. For example, a candidate linker can be evaluated byincubation with dithiothreitol (DTT), or other reducing agent known inthe art, which mimics the rate of cleavage that would be observed in acell, e.g., a target cell. The candidate linkers can also be evaluatedunder conditions which are selected to mimic blood or serum conditions.In a specific embodiment, candidate linkers are cleaved by at most 10%in the blood. In other embodiments, useful candidate linkers aredegraded at least 2, 4, 10, 20, 50, 70, or 100 times faster in the cell(or under in vitro conditions selected to mimic intracellularconditions) as compared to blood (or under in vitro conditions selectedto mimic extracellular conditions).

In yet other embodiments, phosphate-based cleavable linkers are cleavedby agents that degrade or hydrolyze the phosphate group. An example ofan agent that hydrolyzes phosphate groups in cells are enzymes, such asphosphatases in cells. Examples of phosphate-based cleavable groups are—O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O) (ORk)-O—,—O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—,—O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—,—S—P(O)(Rk)-S—, and —O—P(S)(Rk)-S—, where Rk can be hydrogen or alkyl.Specific embodiments include —O—P(O)(OH)—O—, —O—P(S)(OH)—O—,—O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—,—O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—,—S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, and —O—P(S)(H)—S—. Anotherspecific embodiment is —O—P(O)(OH)—O—. These candidate linkers can beevaluated using methods analogous to those described above.

In other embodiments, the linkers may comprise acid cleavable groups,which are groups that are cleaved under acidic conditions. In someembodiments, acid cleavable groups are cleaved in an acidic environmentwith a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower),or by agents, such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes, can providea cleaving environment for acid cleavable groups. Examples of acidcleavable linking groups include, but are not limited to, hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A specific embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl, pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

In other embodiments, the linkers may comprise ester-based cleavablegroups, which are cleaved by enzymes, such as esterases and amidases incells. Examples of ester-based cleavable groups include, but are notlimited to, esters of alkylene, alkenylene and alkynylene groups. Estercleavable groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidate linkers can be evaluated using methods analogous to thosedescribed above.

In further embodiments, the linkers may comprise peptide-based cleavablegroups, which are cleaved by enzymes, such as peptidases and proteasesin cells. Peptide-based cleavable groups are peptide bonds formedbetween amino acids to yield oligopeptides (e.g., dipeptides,tripeptides etc.) and polypeptides. Peptide-based cleavable groups donot include the amide group (—C(O)NH—). The amide group can be formedbetween any alkylene, alkenylene or alkynylene. A peptide bond is aspecial type of amide bond formed between amino acids to yield peptidesand proteins. The peptide based cleavage group is generally limited tothe peptide bond (i.e., the amide bond) formed between amino acidsyielding peptides and proteins and does not include the entire amidefunctional group. Peptide-based cleavable linking groups have thegeneral formula —NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) arethe side chains of the two adjacent amino acids. These candidates can beevaluated using methods analogous to those described above.

Exemplary linkers that can be employed for attaching ligands,particularly ligands comprising a GalNAc moiety, to the sense strand inthe RNAi constructs of the invention, are shown in Formulas A-K below.

In one embodiment, the linker for attaching a ligand to the 3′ end ofthe sense strand of an RNAi construct of the invention has the followingstructure of Formula A, wherein n is 1 or 2, R=ligand (e.g., moietycontaining 3 to 4 GalNAc units) and R′=3′ end of sense strand of adouble stranded RNA molecule:

In another embodiment, the linker for attaching a ligand to the 3′ endof the sense strand of an RNAi construct of the invention has thefollowing structure of Formula B, wherein n is 1, 2, or 3, R=ligand(e.g., moiety containing 3 to 4 GalNAc units) and R′=3′ end of sensestrand of a double stranded RNA molecule:

In yet another embodiment, the linker for attaching a ligand to the 3′end of the sense strand of an RNAi construct of the invention has thefollowing structure of Formula C, wherein n is 1 or 2, R=ligand (e.g.,moiety containing 3 to 4 GalNAc units), R′=3′ end of sense strand of adouble stranded RNA molecule, and R″═H, alkyl, functionalized alkyl:

In certain embodiments, the linker for attaching a ligand to the 3′ endof the sense strand of an RNAi construct of the invention has thefollowing structure of Formula D, wherein R=ligand (e.g., moietycontaining 3 to 4 GalNAc units) and R′=3′ end of sense strand of adouble stranded RNA molecule:

In certain other embodiments, the linker for attaching a ligand to the3′ end of the sense strand of an RNAi construct of the invention has thefollowing structure of Formula E, wherein R=ligand (e.g., moietycontaining 3 to 4 GalNAc units) and R′=3′ end of sense strand of adouble stranded RNA molecule:

In some embodiments, the linker for attaching a ligand to the 3′ end ofthe sense strand of an RNAi construct of the invention has the followingstructure of Formula F, wherein R=ligand (e.g., moiety containing 3 to 4GalNAc units) and R′=3′ end of sense strand of a double stranded RNAmolecule:

In other embodiments, the linker for attaching a ligand to the 3′ end ofthe sense strand of an RNAi construct of the invention has the followingstructure of Formula G, wherein R=ligand (e.g., moiety containing 3 to 4GalNAc units) and R′=3′ end of sense strand of a double stranded RNAmolecule:

In certain other embodiments, the linker for attaching a ligand to the3′ end of the sense strand of an RNAi construct of the invention has thefollowing structure of Formula H, wherein R=ligand (e.g., moietycontaining 3 to 4 GalNAc units) and R′=3′ end of sense strand of adouble stranded RNA molecule:

In some embodiments, the linker for attaching a ligand to the 3′ end ofthe sense strand of an RNAi construct of the invention has the followingstructure of Formula J, wherein R=ligand (e.g., moiety containing 3 to 4GalNAc units) and R′=3′ end of sense strand of a double stranded RNAmolecule:

In other embodiments, the linker for attaching a ligand to the 3′ end ofthe sense strand of an RNAi construct of the invention has the followingstructure of Formula K, wherein R=ligand (e.g., moiety containing 3 to 4GalNAc units) and R′=3′ end of sense strand of a double stranded RNAmolecule:

Other types of linkers suitable for attaching ligands to the sense orantisense strands in the RNAi constructs of the invention are known inthe art and can include the linkers described in U.S. Pat. Nos.7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551, all of whichare hereby incorporated by reference in their entireties.

In certain embodiments, the ligand covalently attached to the sense orantisense strand of the RNAi constructs of the invention comprises aGalNAc moiety, e.g, a multivalent GalNAc moiety. In some embodiments,the multivalent GalNAc moiety is a trivalent GalNAc moiety and isattached to the 3′ end of the sense strand. In other embodiments, themultivalent GalNAc moiety is a trivalent GalNAc moiety and is attachedto the 5′ end of the sense strand. In yet other embodiments, themultivalent GalNAc moiety is a tetravalent GalNAc moiety and is attachedto the 3′ end of the sense strand. In still other embodiments, themultivalent GalNAc moiety is a tetravalent GalNAc moiety and is attachedto the 5′ end of the sense strand. Exemplary trivalent and tetravalentGalNAc moieties and linkers that can be attached to the double-strandedRNA molecules in the RNAi constructs of the invention are provided inthe structural formulas I-XXIX below.

In one embodiment, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula I, wherein each n isindependently 1 to 3, k is 1 to 3, m is 1 or 2, j is 1 or 2, and theligand is attached to the 3′ end of the sense strand of thedouble-stranded RNA molecule (represented by the solid wavy line):

In another embodiment, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula II, wherein each n isindependently 1 to 3, k is 1 to 3, m is 1 or 2, and the ligand isattached to the 3′ end of the sense strand of the double-stranded RNAmolecule (represented by the solid wavy line):

In yet another embodiment, the RNAi construct comprises a ligand andlinker having the following structure of Formula III, wherein each n isindependently 1 to 3, k is 1 to 3, m is 1 or 2, j is 1 or 2, and theligand is attached to the 3′ end of the sense strand of thedouble-stranded RNA molecule (represented by the solid wavy line):

In still another embodiment, the RNAi construct comprises a ligand andlinker having the following structure of Formula IV, wherein each n isindependently 1 to 3, k is 1 to 3, m is 1 or 2, j is 1 or 2, and theligand is attached to the 3′ end of the sense strand of thedouble-stranded RNA molecule (represented by the solid wavy line):

In still another embodiment, the RNAi construct comprises a ligand andlinker having the following structure of Formula V, wherein each n isindependently 1 to 3, k is 1 to 3, m is 1 or 2, j is 1 or 2, and theligand is attached to the 3′ end of the sense strand of thedouble-stranded RNA molecule (represented by the solid wavy line):

In another embodiment, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula VI, wherein each n isindependently 1 to 3, k is 1 to 3, m is 1 or 2, j is 1 or 2, and theligand is attached to the 3′ end of the sense strand of thedouble-stranded RNA molecule (represented by the solid wavy line):

In one particular embodiment, the RNAi construct comprises a ligand andlinker having the following structure of Formula VII, wherein the ligandis attached to the 3′ end of the sense strand of the double-stranded RNAmolecule (represented by the solid wavy line):

In another particular embodiment, the RNAi construct comprises a ligandand linker having the following structure of Formula VIII, wherein theligand is attached to the 3′ end of the sense strand of thedouble-stranded RNA molecule (represented by the solid wavy line):

In certain embodiments, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula IX, wherein each n isindependently 1 to 3, k is 1 to 3, m is 1 or 2, and the ligand isattached to the 3′ end of the sense strand of the double-stranded RNAmolecule (represented by the solid wavy line):

In other embodiments, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula X, wherein each n isindependently 1 to 3, k is 1 to 3, m is 1 or 2, and the ligand isattached to the 3′ end of the sense strand of the double-stranded RNAmolecule (represented by the solid wavy line):

In one embodiment, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XI, wherein each n isindependently 1 to 3, k is 1 to 3, m is 1 or 2, and the ligand isattached to the 3′ end of the sense strand of the double-stranded RNAmolecule (represented by the solid wavy line):

In another embodiment, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XII, wherein each n isindependently 1 to 3, k is 1 to 3, m is 1 or 2, and the ligand isattached to the 3′ end of the sense strand of the double-stranded RNAmolecule (represented by the solid wavy line):

In yet another embodiment, the RNAi construct comprises a ligand andlinker having the following structure of Formula XIII, wherein each n isindependently 1 to 3, k is 1 to 3, m is 1 or 2, and the ligand isattached to the 3′ end of the sense strand of the double-stranded RNAmolecule (represented by the solid wavy line):

In certain embodiments, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XIV, wherein each n isindependently 1 to 3, k is 1 to 3, and the ligand is attached to the 5′end of the sense strand of the double-stranded RNA molecule (representedby the solid wavy line):

In one embodiment, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XV, wherein each n isindependently 1 to 3 and the ligand is attached to the 5′ end of thesense strand of the double-stranded RNA molecule (represented by thesolid wavy line):

In other embodiments, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XVI, wherein each n isindependently 1 to 3, k is 1 to 3, and the ligand is attached to the 5′end of the sense strand of the double-stranded RNA molecule (representedby the solid wavy line):

In one embodiment, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XVII, wherein each n isindependently 1 to 3 and the ligand is attached to the 5′ end of thesense strand of the double-stranded RNA molecule (represented by thesolid wavy line):

In certain other embodiments, the RNAi construct comprises a ligand andlinker having the following structure of Formula XVIII, wherein each nis independently 1 to 3, k is 1 to 3, and the ligand is attached to the5′ end of the sense strand of the double-stranded RNA molecule(represented by the solid wavy line):

In one particular embodiment, the RNAi construct comprises a ligand andlinker having the following structure of Formula XIX, wherein each n isindependently 1 to 3 and the ligand is attached to the 5′ end of thesense strand of the double-stranded RNA molecule (represented by thesolid wavy line):

In some embodiments, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XX, wherein each n isindependently 1 to 3, k is 1 to 3, and the ligand is attached to the 5′end of the sense strand of the double-stranded RNA molecule (representedby the solid wavy line):

In one embodiment, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XXI, wherein each n isindependently 1 to 3, and the ligand is attached to the 5′ end of thesense strand of the double-stranded RNA molecule (represented by thesolid wavy line):

In certain embodiments, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XXII, wherein each n isindependently 1 to 3, k is 1 to 3, and the ligand is attached to the 5′end of the sense strand of the double-stranded RNA molecule (representedby the solid wavy line):

In one embodiment, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XXIII, wherein each n isindependently 1 to 3 and the ligand is attached to the 5′ end of thesense strand of the double-stranded RNA molecule (represented by thesolid wavy line):

In certain other embodiments, the RNAi construct comprises a ligand andlinker having the following structure of Formula XXIV, wherein each n isindependently 1 to 3 and the ligand is attached to the 5′ end of thesense strand of the double-stranded RNA molecule (represented by thesolid wavy line):

In another embodiment, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XXV, wherein each n isindependently 1 to 3 and the ligand is attached to the 5′ end of thesense strand of the double-stranded RNA molecule (represented by thesolid wavy line):

In certain embodiments, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XXVI, wherein the ligand isattached to the 5′ end of the sense strand of the double-stranded RNAmolecule (represented by the solid wavy line):

In one embodiment, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XXVII, wherein each n isindependently 1 to 3, k is 1 to 9, m is 1 or 2, and the ligand isattached to the 5′ end of the sense strand of the double-stranded RNAmolecule (represented by the solid wavy line):

In another embodiment, the RNAi construct comprises a ligand and linkerhaving the following structure of Formula XXVIII, wherein the ligand isattached to the 5′ end of the sense strand of the double-stranded RNAmolecule (represented by the solid wavy line):

In one particular embodiment, the RNAi construct comprises a ligand andlinker having the following structure of Formula XXIX, wherein theligand is attached to the 3′ end of the sense strand of thedouble-stranded RNA molecule (represented by the solid wavy line):

In some embodiments, the RNAi constructs of the invention may bedelivered to a cell or tissue of interest by administering a vector thatencodes and controls the intracellular expression of the RNAi construct.A “vector” (also referred to herein as an “expression vector) is acomposition of matter which can be used to deliver a nucleic acid ofinterest to the interior of a cell. Numerous vectors are known in theart including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “vector” includes an autonomouslyreplicating plasmid or a virus. Examples of viral vectors include, butare not limited to, adenoviral vectors, adeno-associated viral vectors,retroviral vectors, and the like. A vector can be replicated in a livingcell, or it can be made synthetically.

Generally, a vector for expressing an RNAi construct of the inventionwill comprise one or more promoters operably linked to sequencesencoding the RNAi construct. The phrase “operably linked” or “undertranscriptional control” as used herein means that the promoter is inthe correct location and orientation in relation to a polynucleotidesequence to control the initiation of transcription by RNA polymeraseand expression of the polynucleotide sequence. A “promoter” refers to asequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene sequence. Suitable promoters include, but arenot limited to, RNA pol I, pol II, H1 or U6 RNA pol III, and viralpromoters (e.g. human cytomegalovirus (CMV) immediate early genepromoter, the SV40 early promoter, and the Rous sarcoma virus longterminal repeat). In some embodiments, a H1 or U6 RNA pol III promoteris preferred. The promoter can be a tissue-specific or induciblepromoter. Of particular interest are liver-specific promoters, such aspromoter sequences from human alpha 1-antitrypsin gene, albumin gene,hemopexin gene, and hepatic lipase gene. Inducible promoters includepromoters regulated by ecdysone, estrogen, progesterone, tetracycline,and isopropyl-P-D1-thiogalactopyranoside (IPTG).

In some embodiments in which the RNAi construct comprises a siRNA, thetwo separate strands (sense and antisense strand) can be expressed froma single vector or two separate vectors. For example, in one embodiment,the sequence encoding the sense strand is operably linked to a promoteron a first vector and the sequence encoding the antisense strand isoperably linked to a promoter on a second vector. In such an embodiment,the first and second vectors are co-introduced, e.g., by infection ortransfection, into a target cell, such that the sense and antisensestrands, once transcribed, will hybridize intracellularly to form thesiRNA molecule. In another embodiment, the sense and antisense strandsare transcribed from two separate promoters located in a single vector.In some such embodiments, the sequence encoding the sense strand isoperably linked to a first promoter and the sequence encoding theantisense strand is operably linked to a second promoter, wherein thefirst and second promoters are located in a single vector. In oneembodiment, the vector comprises a first promoter operably linked to asequence encoding the siRNA molecule, and a second promoter operablylinked to the same sequence in the opposite direction, such thattranscription of the sequence from the first promoter results in thesynthesis of the sense strand of the siRNA molecule and transcription ofthe sequence from the second promoter results in synthesis of theantisense strand of the siRNA molecule.

In other embodiments in which the RNAi construct comprises a shRNA, asequence encoding the single, at least partially self-complementary RNAmolecule is operably linked to a promoter to produce a singletranscript. In some embodiments, the sequence encoding the shRNAcomprises an inverted repeat joined by a linker polynucleotide sequenceto produce the the stem and loop structure of the shRNA followingtranscription.

In some embodiments, the vector encoding an RNAi construct of theinvention is a viral vector. Various viral vector systems that aresuitable to express the RNAi constructs described herein include, butare not limited to, adenoviral vectors, retroviral vectors (e.g.,lentiviral vectors, moloney murine leukemia virus), adeno-associatedviral vectors; herpes simplex viral vectors; SV40 vectors; polyoma viralvectors; papilloma viral vectors; picornaviral vectors; and pox viralvectors (e.g. vaccinia virus). In certain embodiments, the viral vectoris a retroviral vector (e.g. lentiviral vector).

Various vectors suitable for use in the invention, methods for insertingnucleic acid sequences encoding siRNA or shRNA molecules into vectors,and methods of delivering the vectors to the cells of interest arewithin the skill of those in the art. See, e.g., Dornburg, Gene Therap.,Vol. 2: 301-310, 1995; Eglitis, Biotechniques, Vol. 6: 608-614, 1988;Miller, Hum Gene Therap., Vol. 1: 5-14, 1990; Anderson, Nature, Vol.392: 25-30, 1998; Rubinson D A et al., Nat. Genet., Vol. 33: 401-406,2003; Brummelkamp et al., Science, Vol. 296: 550-553, 2002; Brummelkampet al., Cancer Cell, Vol. 2: 243-247, 2002; Lee et al., Nat Biotechnol,Vol. 20: 500-505, 2002; Miyagishi et al., Nat Biotechnol, Vol. 20:497-500, 2002; Paddison et al., Genes Dev, Vol. 16: 948-958, 2002; Paulet al., Nat Biotechnol, Vol. 20: 505-508, 2002; Sui et al., Proc NatlAcad Sci USA, Vol. 99: 5515-5520, 2002; and Yu et al., Proc Natl AcadSci USA, Vol. 99: 6047-6052, 2002, all of which are hereby incorporatedby reference in their entireties.

The present invention also includes pharmaceutical compositions andformulations comprising the RNAi constructs described herein andpharmaceutically acceptable carriers, excipients, or diluents. Suchcompositions and formulations are useful for reducing expression ofASGR1 in a subject in need thereof. Where clinical applications arecontemplated, pharmaceutical compositions and formulations will beprepared in a form appropriate for the intended application. Generally,this will entail preparing compositions that are essentially free ofpyrogens, as well as other impurities that could be harmful to humans oranimals.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human. As used herein, “pharmaceutically acceptablecarrier, excipient, or diluent” includes solvents, buffers, solutions,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like acceptable for usein formulating pharmaceuticals, such as pharmaceuticals suitable foradministration to humans. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with the RNAiconstructs of the present invention, its use in therapeutic compositionsis contemplated. Supplementary active ingredients also can beincorporated into the compositions, provided they do not inactivate thevectors or RNAi constructs of the compositions.

Compositions and methods for the formulation of pharmaceuticalcompositions depend on a number of criteria, including, but not limitedto, route of administration, type and extent of disease or disorder tobe treated, or dose to be administered. In some embodiments, thepharmaceutical compositions are formulated based on the intended routeof delivery. For instance, in certain embodiments, the pharmaceuticalcompositions are formulated for parenteral delivery. Parenteral forms ofdelivery include intravenous, intraarterial, subcutaneous, intrathecal,intraperitoneal or intramuscular injection or infusion. In oneembodiment, the pharmaceutical composition is formulated for intravenousdelivery. In such an embodiment, the pharmaceutical composition mayinclude a lipid-based delivery vehicle. In another embodiment, thepharmaceutical composition is formulated for subcutaneous delivery. Insuch an embodiment, the pharmaceutical composition may include atargeting ligand (e.g. GalNAc-containing or antibody-containing ligandsdescribed herein).

In some embodiments, the pharmaceutical compositions comprise aneffective amount of an RNAi construct described herein. An “effectiveamount” is an amount sufficient to produce a beneficial or desiredclinical result. In some embodiments, an effective amount is an amountsufficient to reduce ASGR1 expression in hepatocytes of a subject. Insome embodiments, an effective amount may be an amount sufficient toonly partially reduce ASGR1 expression, for example, to a levelcomparable to expression of the wild-type ASGR1 allele in humanheterozygotes. Human heterozygous carriers of loss of function ASGR1variant alleles were reported to have lower serum levels of non-HDLcholesterol and a lower risk of coronary artery disease and myocardialinfarction as compared to non-carriers (Nioi et al., New England Journalof Medicine, Vol. 374(22):2131-2141, 2016). Thus, without being bound bytheory, it is believed that partial reduction of ASGR1 expression may besufficient to achieve the benefical reduction of serum non-HDLcholesterol and reduction of risk of coronary artery disease andmyocardial infarction.

An effective amount of an RNAi construct of the invention may be fromabout 0.01 mg/kg body weight to about 100 mg/kg body weight, about 0.05mg/kg body weight to about 75 mg/kg body weight, about 0.1 mg/kg bodyweight to about 50 mg/kg body weight, about 1 mg/kg to about 30 mg/kgbody weight, about 2.5 mg/kg of body weight to about 20 mg/kg bodyweight, or about 5 mg/kg body weight to about 15 mg/kg body weight. Incertain embodiments, a single effective dose of an RNAi construct of theinvention may be about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg,about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10 mg/kg. Thepharmaceutical composition comprising an effective amount of RNAiconstruct can be administered weekly, biweekly, monthly, quarterly, orbiannually. The precise determination of what would be considered aneffective amount and frequency of administration may be based on severalfactors, including a patient's size, age, and general condition, type ofdisorder to be treated (e.g. myocardial infarction, heart failure,coronary artery disease, hypercholesterolemia), particular RNAiconstruct employed, and route of administration. Estimates of effectivedosages and in vivo half-lives for any particular RNAi construct of theinvention can be ascertained using conventional methods and/or testingin appropriate animal models.

Administration of the pharmaceutical compositions of the presentinvention may be via any common route so long as the target tissue isavailable via that route. Such routes include, but are not limited to,parenteral (e.g., subcutaneous, intramuscular, intraperitoneal orintravenous), oral, nasal, buccal, intradermal, transdermal, andsublingual routes, or by direct injection into liver tissue or deliverythrough the hepatic portal vein. In some embodiments, the pharmaceuticalcomposition is administered parenterally. For instance, in certainembodiments, the pharmaceutical composition is administeredintravenously. In other embodiments, the pharmaceutical composition isadministered subcutaneously.

Colloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems, includingoil-in-water emulsions, micelles, mixed micelles, and liposomes, may beused as delivery vehicles for the RNAi constructs of the invention orvectors encoding such constructs. Commercially available fat emulsionsthat are suitable for delivering the nucleic acids of the inventioninclude Intralipid® (Baxter International Inc.), Liposyn® (AbbottPharmaceuticals), Liposyn II (Hospira), Liposyn III (Hospira),Nutrilipid (B. Braun Medical Inc.), and other similar lipid emulsions. Apreferred colloidal system for use as a delivery vehicle in vivo is aliposome (i.e., an artificial membrane vesicle). The RNAi constructs ofthe invention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively, RNAiconstructs of the invention may be complexed to lipids, in particular tocationic lipids. Suitable lipids and liposomes include neutral (e.g.,dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and dipalmitoyl phosphatidylcholine (DPPC)),distearolyphosphatidyl choline), negative (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl(DOTAP) and dioleoylphosphatidyl ethanolamine (DOTMA)). The preparationand use of such colloidal disperson systems is well known in the art.Exemplary formulations are also disclosed in U.S. Pat. Nos. 5,981,505;6,217,900; 6,383,512; 5,783,565; 7,202,227; 6,379,965; 6,127,170;5,837,533; 6,747,014; and WO03/093449.

In some embodiments, the RNAi constructs of the invention are fullyencapsulated in a lipid formulation, e.g., to form a SPLP, pSPLP, SNALP,or other nucleic acid-lipid particle. As used herein, the term “SNALP”refers to a stable nucleic acid-lipid particle, including SPLP. As usedherein, the term “SPLP” refers to a nucleic acid-lipid particlecomprising plasmid DNA encapsulated within a lipid vesicle. SNALPs andSPLPs typically contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). SNALPs and SPLPs are exceptionally useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). SPLPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The nucleicacid-lipid particles typically have a mean diameter of about 50 nm toabout 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm,or about 70 nm to about 90 nm, and are substantially nontoxic. Inaddition, the nucleic acids when present in the nucleic acid-lipidparticles are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

The pharmaceutical compositions suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. Generally, these preparations are sterile and fluid to theextent that easy injectability exists. Preparations should be stableunder the conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. Appropriate solvents or dispersion media may contain, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompounds in an appropriate amount into a solvent along with any otheringredients (for example as enumerated above) as desired, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the desired otheringredients, e.g., as enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation include vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient(s) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions of the present invention generally may be formulated ina neutral or salt form. Pharmaceutically-acceptable salts include, forexample, acid addition salts (formed with free amino groups) derivedfrom inorganic acids (e.g., hydrochloric or phosphoric acids), or fromorganic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like).Salts formed with the free carboxyl groups can also be derived frominorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferrichydroxides) or from organic bases (e.g., isopropylamine, trimethylamine,histidine, procaine and the like).

For parenteral administration in an aqueous solution, for example, thesolution generally is suitably buffered and the liquid diluent firstrendered isotonic for example with sufficient saline or glucose. Suchaqueous solutions may be used, for example, for intravenous,intramuscular, subcutaneous and intraperitoneal administration.Preferably, sterile aqueous media are employed as is known to those ofskill in the art, particularly in light of the present disclosure. Byway of illustration, a single dose may be dissolved in 1 ml of isotonicNaCl solution and either added to 1000 ml of hypodermoclysis fluid orinjected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). For human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA standards. In certain embodiments, a pharmaceutical compositionof the invention comprises or consists of a sterile saline solution andan RNAi construct described herein. In other embodiments, apharmaceutical composition of the invention comprises or consists of anRNAi construct described herein and sterile water (e.g. water forinjection, WFI). In still other embodiments, a pharmaceuticalcomposition of the invention comprises or consists of an RNAi constructdescribed herein and phosphate-buffered saline (PBS).

In some embodiments, the pharmaceutical compositions of the inventionare packaged with or stored within a device for administration. Devicesfor injectable formulations include, but are not limited to, injectionports, pre-filled syringes, autoinjectors, injection pumps, on-bodyinjectors, and injection pens. Devices for aerosolized or powderformulations include, but are not limited to, inhalers, insufflators,aspirators, and the like. Thus, the present invention includesadministration devices comprising a pharmaceutical composition of theinvention for treating or preventing one or more of the disordersdescribed herein.

The present invention provides methods for reducing or inhibitingexpression of ASGR1 in a subject in need thereof as well as methods oftreating or preventing conditions, diseases, or disorders associatedwith ASGR1 expression or activity. A “condition, disease, or disorderassociated with ASGR1 expression” refers to conditions, diseases, ordisorders in which ASGR1 expression levels are altered or where elevatedexpression levels of ASGR1 are associated with an increased risk ofdeveloping the condition, disease or disorder. A condition, disease, ordisorder associated with ASGR1 expression can also include conditions,diseases, or disorders resulting from aberrant changes in lipoproteinmetabolism, such as changes resulting in abnormal levels of cholesterol,lipids, triglycerides, etc. or impaired clearance of these molecules.Recently, human carriers of loss of function variant alleles of theASGR1 subunit of the asialoglycoprotein receptor were reported to havelower serum levels of non-HDL cholesterol and a lower risk of coronaryartery disease and myocardial infarction as compared to non-carriers(Nioi et al., New England Journal of Medicine, Vol. 374(22):2131-2141,2016, which is hereby incorporated by reference in its entirety). Thus,in certain embodiments, the RNAi constructs of the invention areparticularly useful for treating or preventing cardiovascular disease(e.g. coronary artery disease and myocardial infarction) andcholesterol-related disorders (e.g. hypercholesterolemia).

Conditions, diseases, and disorders associated with ASGR1 expressionthat can be treated or prevented according to the methods of theinvention include, but are not limited to, cardiovascular disease, suchas myocardial infarction, heart failure, stroke (ischemic andhemorrhagic), atherosclerosis, coronary artery disease, peripheralvascular disease (e.g. peripheral artery disease), vulnerable plaque,hypercholesterolemia, and dyslipidemia (manifesting, e.g., as elevatedtotal cholesterol, elevated low-density lipoprotein (LDL), elevated verylow-density lipoprotein (VLDL), elevated triglycerides, and/or lowlevels of high-density lipoprotein (HDL)).

In certain embodiments, the present invention provides a method forreducing the expression of ASGR1 in a patient in need thereof comprisingadministering to the patient any of the RNAi constructs describedherein. The term “patient,” as used herein, refers to a mammal,including humans, and can be used interchangeably with the term“subject.” Preferably, the expression level of ASGR1 in hepatocytes inthe patient is reduced following administration of the RNAi construct ascompared to the ASGR1 expression level in a patient not receiving theRNAi construct.

In some embodiments, a patient in need of reduction of ASGR1 expressionis a patient who is at risk of having a myocardial infarction. A patientwho is at risk of having a myocardial infarction may be a patient whohas a history of myocardial infarction (e.g. has had a previousmyocardial infarction). A patient at risk of having a myocardialinfarction may also be a patient who has a familial history ofmyocardial infarction or who has one or more risk factors of myocardialinfarction. Such risk factors include, but are not limited to,hypertension, elevated levels of non-HDL cholesterol, elevated levels oftriglycerides, diabetes, obesity, or history of autoimmune diseases(e.g. rheumatoid arthritis, lupus). In one embodiment, a patient who isat risk of having a myocardial infarction is a patient who has or isdiagnosed with coronary artery disease. The risk of myocardialinfarction in these and other patients can be reduced by administeringto the patients any of the RNAi constructs described herein.Accordingly, the present invention provides a method for reducing therisk of myocardial infarction in a patient in need thereof comprisingadministering to the patient an RNAi construct described herein. In someembodiments, the present invention includes use of any of the RNAiconstructs described herein in the preparation of a medicament forreducing the risk of myocardial infarction in a patient in need thereof.In other embodiments, the present invention provides an ASGR1-targetingRNAi construct for use in a method for reducing the risk of myocardialinfarction in a patient in need thereof.

In certain embodiments, a patient in need of reduction of ASGR1expression is a patient who is diagnosed with or at risk ofcardiovascular disease. Thus, the present invention includes a methodfor treating or preventing cardiovascular disease in a patient in needthereof by administering any of the RNAi constructs of the invention. Insome embodiments, the present invention includes use of any of the RNAiconstructs described herein in the preparation of a medicament fortreating or preventing cardiovascular disease in a patient in needthereof. In other embodiments, the present invention provides anASGR1-targeting RNAi construct for use in a method for treating orpreventing cardiovascular disease in a patient in need thereof.Cardiovascular disease includes myocardial infarction, heart failure,stroke (ischemic and hemorrhagic), atherosclerosis, coronary arterydisease, peripheral vascular disease (e.g. peripheral artery disease),and vulnerable plaque. In some embodiments, the cardiovascular diseaseto be treated or prevented according to the methods of the invention iscoronary artery disease. In other embodiments, the cardiovasculardisease to be treated or prevented according to the methods of theinvention is myocardial infarction. In certain embodiments,administration of the RNAi constructs described herein reduces the riskof non-fatal myocardial infarctions, fatal and non-fatal strokes,certain types of heart surgery (e.g. angioplasty, bypass),hospitalization for heart failure, chest pain in patients with heartdisease, and/or cardiovascular events in patients with established heartdisease (e.g. prior myocardial infarction, prior heart surgery, and/orchest pain with evidence of blocked arteries). In some embodiments,administration of the RNAi constructs described herein according to themethods of the invention can be used to reduce the risk of recurrentcardiovascular events.

In certain other embodiments, a patient in need of reduction of ASGR1expression is a patient who has elevated levels of non-HDL cholesterol.Accordingly, in some embodiments, the present invention provides amethod for reducing non-HDL cholesterol in a patient in need thereof byadministering to the patient any of the RNAi constructs describedherein. In some embodiments, the present invention includes use of anyof the RNAi constructs described herein in the preparation of amedicament for reducing non-HDL cholesterol in a patient in needthereof. In other embodiments, the present invention provides anASGR1-targeting RNAi construct for use in a method for reducing non-HDLcholesterol in a patient in need thereof. Non-HDL cholesterol is ameasure of all cholesterol-containing proatherogenic lipoproteins,including LDL cholesterol, very low-density lipoprotein,intermediate-density lipoprotein, lipoprotein (a), chylomicron, andchylomicron remnants. Non-HDL cholesterol has been reported to be a goodpredictor of cardiovascular risk (Rana et al., Curr. Atheroscler. Rep.,Vol. 14:130-134, 2012). Non-HDL cholesterol levels can be calculated bysubtracting HDL cholesterol levels from total cholesterol levels. In oneembodiment, a patient's LDL cholesterol levels are reduced followingadministration of the RNAi construct. In another embodiment, a patient'slipoprotein (a) levels are reduced following administration of the RNAiconstruct.

In some embodiments, a patient to be treated according to the methods ofthe invention is a patient who has elevated levels of non-HDLcholesterol (e.g. elevated serum levels of non-HDL cholesterol).Ideally, levels of non-HDL cholesterol should be about 30 mg/dL abovethe target for LDL cholesterol levels for any given patient. Inparticular embodiments, a patient is administered an RNAi construct ofthe invention if the patient has a non-HDL cholesterol level of about130 mg/dL or greater. In one embodiment, a patient is administered anRNAi construct of the invention if the patient has a non-HDL cholesterollevel of about 160 mg/dL or greater. In another embodiment, a patient isadministered an RNAi construct of the invention if the patient has anon-HDL cholesterol level of about 190 mg/dL or greater. In stillanother embodiment, a patient is administered an RNAi construct of theinvention if the patient has a non-HDL cholesterol level of about 220mg/dL or greater. In certain embodiments, a patient is administered anRNAi construct of the invention if the patient is at a high or very highrisk of cardiovascular disease according to the 2013 ACC/AHA Guidelineon the Assessment of Cardiovascular Risk (Goff et al., ACC/AHA guidelineon the assessment of cardiovascular risk: a report of the AmericanCollege of Cardiology/American Heart Association Task Force on PracticeGuidelines. Circulation. 2013; 00:000-000) and has a non-HDL cholesterollevel of about 100 mg/dL or greater.

In some embodiments of the methods of the invention, a patient isadministered an RNAi construct described herein if they are at amoderate risk or higher for cardiovascular disease according to the 2013ACC/AHA Guideline on the Assessment of Cardiovascular Risk (referred toherein as the “2013 Guidelines”). In certain embodiments, an RNAiconstruct of the invention is administered to a patient if the patient'sLDL cholesterol level is greater than about 160 mg/dL. In otherembodiments, an RNAi construct of the invention is administered to apatient if the patient's LDL cholesterol level is greater than about 130mg/dL and the patient has a moderate risk of cardiovascular diseaseaccording to the 2013 Guidelines. In still other embodiments, an RNAiconstruct of the invention is administered to a patient if the patient'sLDL cholesterol level is greater than 100 mg/dL and the patient has ahigh or very high risk of cardiovascular disease according to the 2013Guidelines.

In certain embodiments, a patient to be treated according to the methodsof the invention is a patient who has a vulnerable plaque (also referredto as unstable plaque). Vulnerable plaques are a build-up of macrophagesand lipids containing predominantly cholesterol that lie underneath theendothelial lining of the arterial wall. These vulnerable plaques canrupture resulting in the formation of a blood clot, which canpotentially block blood flow through the artery and cause a myocardialinfarction or stroke. Vulnerable plaques can be identified by methodsknown in the art, including, but not limited to, intravascularultrasound and computed tomography (Sahara et al., European HeartJournal, Vol. 25: 2026-2033, 2004; Budhoff, J. Am. Coll. Cardiol., Vol.48: 319-321, 2006; Hausleiter et al., J. Am. Coll. Cardiol., Vol. 48:312-318, 2006).

In some embodiments of the methods of the invention, the RNAi constructis administered in combination with another therapeutic agent, such as atherapeutic agent for treating or preventing cardiovascular disease. Inone embodiment, an RNAi construct of the invention is administered aloneor in combination with other agents useful for treating the conditionwith which the patient is afflicted. Examples of such agents includeboth proteinaceous and non-proteinaceous drugs. When multipletherapeutics are co-administered, dosages may be adjusted accordingly,as is recognized in the pertinent art. “Co-administration” andcombination therapy are not limited to simultaneous administration, butalso include treatment regimens in which an RNAi construct of theinvention is administered at least once during a course of treatmentthat involves administering at least one other therapeutic agent to thepatient. In certain embodiments, an RNAi construct of the invention isadministered prior to the administration of at least one othertherapeutic agent. In other embodiments, an RNAi construct of theinvention is administered concurrent with the administration of at leastone other therapeutic agent. In some embodiments, an RNAi construct ofthe invention is administered subsequent to the administration of atleast one other therapeutic agent.

In certain embodiments of the methods of the invention, the RNAiconstruct is administered to a patient in combination with a PCSK9antagonist, such as an anti-hPCSK9 antibody (e.g., Repatha®(evolocumab)). In another embodiment, the RNAi construct of theinvention is administered to a patient in combination with at least oneother cholesterol-lowering (serum and/or total body cholesterol) agent.In some embodiments, the agent increases the expression of LDLR, hasbeen observed to increase serum HDL levels, lower LDL levels, or lowertriglyceride levels. Exemplary agents include, but are not limited to,statins (e.g., atorvastatin, cerivastatin, fluvastatin, lovastatin,mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin);nicotinic acid (Niacin) (NIACOR, NIASPAN (slow release niacin),SLO-NIACIN (slow release niacin)); fibric acid (LOPID (Gemfibrozil),TRICOR (fenofibrate)); bile acid sequestrants (QUESTRAN(cholestyramine), colesevelam (WELCHOL), COLESTID (colestipol));cholesterol absorption inhibitors (ZETIA (ezetimibe)), combiningnicotinic acid with statin (ADVICOR (LOVASTATIN and NIASPAN),combinations of a statin with an absorption inhibitor (VYTORIN (ZOCORand ZETIA); and/or lipid modifying agents.

In some embodiments, the RNAi construct of the invention is combinedwith PPAR gamma agonists, PPAR alpha/gamma agonists, squalene synthaseinhibitors, CETP inhibitors, anti-hypertensives, anti-diabetic agents(such as sulphonyl ureas, insulin, GLP-1 analogs, DDPIV inhibitors),ApoB modulators, MTP inhibitors and/or arteriosclerosis obliteranstreatments. In certain embodiments, the RNAi construct of the inventionis combined with an agent that increases the level of LDL receptor(LDLR) protein in a patient, such as statins, certain cytokines, likeoncostatin M, estrogen, and/or certain herbal ingredients, such asberberine.

In some embodiments, the RNAi construct of the invention is combinedwith an agent that increases serum cholesterol levels in a patient (suchas certain anti-psycotic agents, certain HIV protease inhibitors,dietary factors such as high fructose, sucrose, cholesterol or certainfatty acids and certain nuclear receptor agonists and antagonists forRXR, RAR, LXR, FXR). In certain embodiments, the RNAi construct of theinvention is combined with an agent that increases the level of PCSK9protein in a subject, such as statins and/or insulin. The administrationof RNAi constructs in such embodiments can allow for the RNAi constructto mitigate the undesirable side-effects of these other agents, such asincreases in serum non-HDL cholesterol.

It is understood that all ribonucleic acid sequences disclosed hereincan be converted to deoxyribonucleic acid sequences by substituting athymine base for a uracil base in the sequence. Likewise, alldeoxyribonucleic acid sequences disclosed herein can be converted toribonucleic acid sequences by substituting a uracil base for a thyminebase in the sequence. Deoxyribonucleic acid sequences, ribonucleic acidsequences, and sequences containing mixtures of deoxyribonucleotides andribonucleotides of all sequences disclosed herein are included in theinvention.

Additionally, any nucleic acid sequences disclosed herein may bemodified with any combination of chemical modifications. One of skill inthe art will readily appreciate that such designation as “RNA” or “DNA”to describe modified polynucleotides is, in certain instances,arbitrary. For example, a polynucleotide comprising a nucleotide havinga 2′-OH substituent on the ribose sugar and a thymine base could bedescribed as a DNA molecule having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA molecule having a modified base(thymine (methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of a further example and withoutlimitation, a polynucleotide having the sequence “ATCGATCG” encompassesany polynucleotides having such a sequence, whether modified orunmodified, including, but not limited to, such compounds comprising RNAbases, such as those having sequence “AUCGAUCG” and those having someDNA bases and some RNA bases such as “AUCGATCG” and polynucleotideshaving other modified bases, such as “ATmeCGAUCG,” wherein meC indicatesa cytosine base comprising a methyl group at the 5-position.

The following examples, including the experiments conducted and theresults achieved, are provided for illustrative purposes only and arenot to be construed as limiting the scope of the appended claims.

EXAMPLES Example 1. Selection and Design of ASGR1 siRNA Sequences

The identification and selection of optimal sequences for therapeuticsiRNA molecules targeting the human asialoglycoprotein receptor 1(ASGR1) proceeded in two phases. Candidate sequences for inclusion in aninitial Tier 1 screening set were identified using a bioinformaticsanalysis of two alternatively spliced transcripts for human ASGR1:transcript variant 1 encoding the longer isoform A (NCBI ReferenceSequence No. NM_001671.4; see FIG. 1A) and transcript variant 2 encodingthe shorter isoform B (NCBI Reference Sequence No. NM_001197216.2; seeFIG. 1B). The two human ASGR1 transcript sequences were analyzed usingan in-house siRNA design algorithm, which identifies 19mer sequenceshaving a particular base content at certain positions or regions withinthe 19mer sequences. Sequences were also evaluated for identity to ASGR1sequences in the mouse (NCBI Reference No. NM_009714.2; see FIG. 2), rat(FIG. 3), and cynomolgus monkey (NCBI Reference No. XM_005582698.1; seeFIG. 4). 19mer sequences were also evaluated for sequence identity toother human gene sequences to predict off-target effects and for overlapwith known single nucleotide polymorphisms. Based on the results of thebioinformatics analysis, 211 sequences were included in the Tier 1screening set. A UU dinucleotide was added to the 3′ end of the selected19mer sense and antisense sequences to produce siRNA molecules with 19base pair duplex regions and a 2 nucleotide overhang at the 3′ ends ofboth strands.

The second phase of siRNA sequence selection was directed to identifyingadditional active siRNAs targeting the human ASGR1 mRNA that may havebeen excluded as a result of the bioinformatics analysis. Alloverlapping 19mer sequences from the human ASGR1 transcript variants 1and 2 (NCBI Reference Sequence Nos. NM_001671.4 and NM_001197216.2; seeFIGS. 1A and 1B) were extracted and reverse complement antisensesequences designed. 1284 sequences that were not included in Tier 1 wereincluded as part of the Tier 2 sequences. Like the Tier 1 sequences, theTier 2 sequences were converted to 21mers by adding a UU dinucleotide tothe 3′ ends of the sense and antisense strand to produce siRNA moleculeswith 19 base pair duplex regions and 2 nucleotide overhangs at each 3′end. Tier 2 also included 55 sequences identified in Tier 1 forresynthesis and testing. The combined Tier 1 and Tier 2 screening setsincluded 1495 19mer sequences across the human ASGR1 mRNA transcripts.The sense and antisense sequences of the siRNA molecules included inboth Tier 1 and Tier 2 screening sets, as well as the sequences for 8additional siRNA molecules that target the terminal regions of thetranscripts, are shown in Table 1 below. The site within each of thehuman ASGR1 transcripts that is targeted by each of the siRNA moleculesis also listed in Table 1.

TABLE 1 ASGR1 siRNA Sequences Target site of Target site SEQ antisenseof antisense SEQ ID sequence sequence ID NO: Duplex within withinSense Sequence NO: Antisense Sequence anti- No. NM_001671.4NM_001197216.2 (5′-3′) sense (5′-3′) sense D-1000 1000-1018 883-901UGUCCAGCACCACAUAGGCUU 5 GCCUAUGUGGUGCUGGACAUU 1508 D-1001 1001-1019884-902 GUCCAGCACCACAUAGGCCUU 6 GGCCUAUGUGGUGCUGGACUU 1509 D-1002100-118 100-118 CACCCCCACACUCCCCUAAUU 7 UUAGGGGAGUGUGGGGGUGUU 1510D-1003 1002-1020 885-903 UCCAGCACCACAUAGGCCCUU 8 GGGCCUAUGUGGUGCUGGAUU1511 D-1004 1003-1021 886-904 CCAGCACCACAUAGGCCCUUU 9AGGGCCUAUGUGGUGCUGGUU 1512 D-1005 1004-1022 887-905CAGCACCACAUAGGCCCUGUU 10 CAGGGCCUAUGUGGUGCUGUU 1513 D-1006 1005-1023888-906 AGCACCACAUAGGCCCUGUUU 11 ACAGGGCCUAUGUGGUGCUUU 1514 D-10071006-1024 889-907 GCACCACAUAGGCCCUGUGUU 12 CACAGGGCCUAUGUGGUGCUU 1515D-1008 1007-1025 890-908 CACCACAUAGGCCCUGUGAUU 13 UCACAGGGCCUAUGUGGUGUU1516 D-1009 1008-1026 891-909 ACCACAUAGGCCCUGUGAAUU 14UUCACAGGGCCUAUGUGGUUU 1517 D-1010 1009-1027 892-910CCACAUAGGCCCUGUGAACUU 15 GUUCACAGGGCCUAUGUGGUU 1518 D-1011 1010-1028893-911 CACAUAGGCCCUGUGAACAUU 16 UGUUCACAGGGCCUAUGUGUU 1519 D-10121011-1029 894-912 ACAUAGGCCCUGUGAACACUU 17 GUGUUCACAGGGCCUAUGUUU 1520D-1013 101-119 101-119 ACCCCCACACUCCCCUAAGUU 18 CUUAGGGGAGUGUGGGGGUUU1521 D-1014 1012-1030 895-913 CAUAGGCCCUGUGAACACCUU 19GGUGUUCACAGGGCCUAUGUU 1522 D-1015 1013-1031 896-914AUAGGCCCUGUGAACACCUUU 20 AGGUGUUCACAGGGCCUAUUU 1523 D-1016 1014-1032897-915 UAGGCCCUGUGAACACCUGUU 21 CAGGUGUUCACAGGGCCUAUU 1524 D-10171015-1033 898-916 AGGCCCUGUGAACACCUGGUU 22 CCAGGUGUUCACAGGGCCUUU 1525D-1018 1016-1034 899-917 GGCCCUGUGAACACCUGGAUU 23 UCCAGGUGUUCACAGGGCCUU1526 D-1019 1017-1035 900-918 GCCCUGUGAACACCUGGAUUU 24AUCCAGGUGUUCACAGGGCUU 1527 D-1020 1018-1036 901-919CCCUGUGAACACCUGGAUGUU 25 CAUCCAGGUGUUCACAGGGUU 1528 D-1021 1019-1037902-920 CCUGUGAACACCUGGAUGGUU 26 CCAUCCAGGUGUUCACAGGUU 1529 D-10221020-1038 903-921 CUGUGAACACCUGGAUGGGUU 27 CCCAUCCAGGUGUUCACAGUU 1530D-1023 1021-1039 904-922 UGUGAACACCUGGAUGGGCUU 28 GCCCAUCCAGGUGUUCACAUU1531 D-1024 102-120 102-120 CCCCCACACUCCCCUAAGUUU 29ACUUAGGGGAGUGUGGGGGUU 1532 D-1025 1022-1040 905-923GUGAACACCUGGAUGGGCCUU 30 GGCCCAUCCAGGUGUUCACUU 1533 D-1026 1023-1041906-924 UGAACACCUGGAUGGGCCUUU 31 AGGCCCAUCCAGGUGUUCAUU 1534 D-10271024-1042 907-925 GAACACCUGGAUGGGCCUCUU 32 GAGGCCCAUCCAGGUGUUCUU 1535D-1028 1025-1043 908-926 AACACCUGGAUGGGCCUCCUU 33 GGAGGCCCAUCCAGGUGUUUU1536 D-1029 1026-1044 909-927 ACACCUGGAUGGGCCUCCAUU 34UGGAGGCCCAUCCAGGUGUUU 1537 D-1030 1027-1045 910-928CACCUGGAUGGGCCUCCACUU 35 GUGGAGGCCCAUCCAGGUGUU 1538 D-1031 10-28 10-28UGCACGGAAGAGUGAGGUGUU 36 CACCUCACUCUUCCGUGCAUU 1539 D-1032 1028-1046911-929 ACCUGGAUGGGCCUCCACGUU 37 CGUGGAGGCCCAUCCAGGUUU 1540 D-10331029-1047 912-930 CCUGGAUGGGCCUCCACGAUU 38 UCGUGGAGGCCCAUCCAGGUU 1541D-1034 1030-1048 913-931 CUGGAUGGGCCUCCACGACUU 39 GUCGUGGAGGCCCAUCCAGUU1542 D-1035 1031-1049 914-932 UGGAUGGGCCUCCACGACCUU 40GGUCGUGGAGGCCCAUCCAUU 1543 D-1036 103-121 103-121 CCCCACACUCCCCUAAGUUUU41 AACUUAGGGGAGUGUGGGGUU 1544 D-1037 1032-1050 915-933GGAUGGGCCUCCACGACCAUU 42 UGGUCGUGGAGGCCCAUCCUU 1545 D-1038 1033-1051916-934 GAUGGGCCUCCACGACCAAUU 43 UUGGUCGUGGAGGCCCAUCUU 1546 D-10391034-1052 917-935 AUGGGCCUCCACGACCAAAUU 44 UUUGGUCGUGGAGGCCCAUUU 1547D-1040 1035-1053 918-936 UGGGCCUCCACGACCAAAAUU 45 UUUUGGUCGUGGAGGCCCAUU1548 D-1041 1036-1054 919-937 GGGCCUCCACGACCAAAACUU 46GUUUUGGUCGUGGAGGCCCUU 1549 D-1042 1037-1055 920-938GGCCUCCACGACCAAAACGUU 47 CGUUUUGGUCGUGGAGGCCUU 1550 D-1043 1038-1056921-939 GCCUCCACGACCAAAACGGUU 48 CCGUUUUGGUCGUGGAGGCUU 1551 D-10441039-1057 922-940 CCUCCACGACCAAAACGGGUU 49 CCCGUUUUGGUCGUGGAGGUU 1552D-1045 1040-1058 923-941 CUCCACGACCAAAACGGGCUU 50 GCCCGUUUUGGUCGUGGAGUU1553 D-1046 1041-1059 924-942 UCCACGACCAAAACGGGCCUU 51GGCCCGUUUUGGUCGUGGAUU 1554 D-1047 104-122 104-122 CCCACACUCCCCUAAGUUCUU52 GAACUUAGGGGAGUGUGGGUU 1555 D-1048 1042-1060 925-943CCACGACCAAAACGGGCCCUU 53 GGGCCCGUUUUGGUCGUGGUU 1556 D-1049 1043-1061926-944 CACGACCAAAACGGGCCCUUU 54 AGGGCCCGUUUUGGUCGUGUU 1557 D-10501044-1062 927-945 ACGACCAAAACGGGCCCUGUU 55 CAGGGCCCGUUUUGGUCGUUU 1558D-1051 1045-1063 928-946 CGACCAAAACGGGCCCUGGUU 56 CCAGGGCCCGUUUUGGUCGUU1559 D-1052 1046-1064 929-947 GACCAAAACGGGCCCUGGAUU 57UCCAGGGCCCGUUUUGGUCUU 1560 D-1053 1047-1065 930-948ACCAAAACGGGCCCUGGAAUU 58 UUCCAGGGCCCGUUUUGGUUU 1561 D-1054 1048-1066931-949 CCAAAACGGGCCCUGGAAGUU 59 CUUCCAGGGCCCGUUUUGGUU 1562 D-10551049-1067 932-950 CAAAACGGGCCCUGGAAGUUU 60 ACUUCCAGGGCCCGUUUUGUU 1563D-1056 1050-1068 933-951 AAAACGGGCCCUGGAAGUGUU 61 CACUUCCAGGGCCCGUUUUUU1564 D-1057 1051-1069 934-952 AAACGGGCCCUGGAAGUGGUU 62CCACUUCCAGGGCCCGUUUUU 1565 D-1058 105-123 105-123 CCACACUCCCCUAAGUUCCUU63 GGAACUUAGGGGAGUGUGGUU 1566 D-1059 1052-1070 935-953AACGGGCCCUGGAAGUGGGUU 64 CCCACUUCCAGGGCCCGUUUU 1567 D-1060 1053-1071936-954 ACGGGCCCUGGAAGUGGGUUU 65 ACCCACUUCCAGGGCCCGUUU 1568 D-10611054-1072 937-955 CGGGCCCUGGAAGUGGGUGUU 66 CACCCACUUCCAGGGCCCGUU 1569D-1062 1055-1073 938-956 GGGCCCUGGAAGUGGGUGGUU 67 CCACCCACUUCCAGGGCCCUU1570 D-1063 1056-1074 939-957 GGCCCUGGAAGUGGGUGGAUU 68UCCACCCACUUCCAGGGCCUU 1571 D-1064 1057-1075 940-958GCCCUGGAAGUGGGUGGACUU 69 GUCCACCCACUUCCAGGGCUU 1572 D-1065 1058-1076941-959 CCCUGGAAGUGGGUGGACGUU 70 CGUCCACCCACUUCCAGGGUU 1573 D-10661059-1077 942-960 CCUGGAAGUGGGUGGACGGUU 71 CCGUCCACCCACUUCCAGGUU 1574D-1067 1060-1078 943-961 CUGGAAGUGGGUGGACGGGUU 72 CCCGUCCACCCACUUCCAGUU1575 D-1068 1061-1079 944-962 UGGAAGUGGGUGGACGGGAUU 73UCCCGUCCACCCACUUCCAUU 1576 D-1069 106-124 106-124 CACACUCCCCUAAGUUCCAUU74 UGGAACUUAGGGGAGUGUGUU 1577 D-1070 1062-1080 945-963GGAAGUGGGUGGACGGGACUU 75 GUCCCGUCCACCCACUUCCUU 1578 D-1071 1063-1081946-964 GAAGUGGGUGGACGGGACGUU 76 CGUCCCGUCCACCCACUUCUU 1579 D-10721064-1082 947-965 AAGUGGGUGGACGGGACGGUU 77 CCGUCCCGUCCACCCACUUUU 1580D-1073 1065-1083 948-966 AGUGGGUGGACGGGACGGAUU 78 UCCGUCCCGUCCACCCACUUU1581 D-1074 1066-1084 949-967 GUGGGUGGACGGGACGGACUU 79GUCCGUCCCGUCCACCCACUU 1582 D-1075 1067-1085 950-968UGGGUGGACGGGACGGACUUU 80 AGUCCGUCCCGUCCACCCAUU 1583 D-1076 1068-1086951-969 GGGUGGACGGGACGGACUAUU 81 UAGUCCGUCCCGUCCACCCUU 1584 D-10771069-1087 952-970 GGUGGACGGGACGGACUACUU 82 GUAGUCCGUCCCGUCCACCUU 1585D-1078 1070-1088 953-971 GUGGACGGGACGGACUACGUU 83 CGUAGUCCGUCCCGUCCACUU1586 D-1079 1071-1089 954-972 UGGACGGGACGGACUACGAUU 84UCGUAGUCCGUCCCGUCCAUU 1587 D-1080 107-125 107-125 ACACUCCCCUAAGUUCCAAUU85 UUGGAACUUAGGGGAGUGUUU 1588 D-1081 1072-1090 955-973GGACGGGACGGACUACGAGUU 86 CUCGUAGUCCGUCCCGUCCUU 1589 D-1082 1073-1091956-974 GACGGGACGGACUACGAGAUU 87 UCUCGUAGUCCGUCCCGUCUU 1590 D-10831074-1092 957-975 ACGGGACGGACUACGAGACUU 88 GUCUCGUAGUCCGUCCCGUUU 1591D-1084 1075-1093 958-976 CGGGACGGACUACGAGACGUU 89 CGUCUCGUAGUCCGUCCCGUU1592 D-1085 1076-1094 959-977 GGGACGGACUACGAGACGGUU 90CCGUCUCGUAGUCCGUCCCUU 1593 D-1086 1077-1095 960-978GGACGGACUACGAGACGGGUU 91 CCCGUCUCGUAGUCCGUCCUU 1594 D-1087 1078-1096961-979 GACGGACUACGAGACGGGCUU 92 GCCCGUCUCGUAGUCCGUCUU 1595 D-10881079-1097 962-980 ACGGACUACGAGACGGGCUUU 93 AGCCCGUCUCGUAGUCCGUUU 1596D-1089 1080-1098 963-981 CGGACUACGAGACGGGCUUUU 94 AAGCCCGUCUCGUAGUCCGUU1597 D-1090 1081-1099 964-982 GGACUACGAGACGGGCUUCUU 95GAAGCCCGUCUCGUAGUCCUU 1598 D-1091 108-126 108-126 CACUCCCCUAAGUUCCAAUUU96 AUUGGAACUUAGGGGAGUGUU 1599 D-1092 1082-1100 965-983GACUACGAGACGGGCUUCAUU 97 UGAAGCCCGUCUCGUAGUCUU 1600 D-1093 1083-1101966-984 ACUACGAGACGGGCUUCAAUU 98 UUGAAGCCCGUCUCGUAGUUU 1601 D-10941084-1102 967-985 CUACGAGACGGGCUUCAAGUU 99 CUUGAAGCCCGUCUCGUAGUU 1602D-1095 1085-1103 968-986 UACGAGACGGGCUUCAAGAUU 100 UCUUGAAGCCCGUCUCGUAUU1603 D-1096 1086-1104 969-987 ACGAGACGGGCUUCAAGAAUU 101UUCUUGAAGCCCGUCUCGUUU 1604 D-1097 1087-1105 970-988CGAGACGGGCUUCAAGAACUU 102 GUUCUUGAAGCCCGUCUCGUU 1605 D-1098 1088-1106971-989 GAGACGGGCUUCAAGAACUUU 103 AGUUCUUGAAGCCCGUCUCUU 1606 D-10991089-1107 972-990 AGACGGGCUUCAAGAACUGUU 104 CAGUUCUUGAAGCCCGUCUUU 1607D-1100 1090-1108 973-991 GACGGGCUUCAAGAACUGGUU 105 CCAGUUCUUGAAGCCCGUCUU1608 D-1101 1091-1109 974-992 ACGGGCUUCAAGAACUGGAUU 106UCCAGUUCUUGAAGCCCGUUU 1609 D-1102 109-127 109-127 ACUCCCCUAAGUUCCAAUCUU107 GAUUGGAACUUAGGGGAGUUU 1610 D-1103 1092-1110 975-993CGGGCUUCAAGAACUGGAGUU 108 CUCCAGUUCUUGAAGCCCGUU 1611 D-1104 1093-1111976-994 GGGCUUCAAGAACUGGAGGUU 109 CCUCCAGUUCUUGAAGCCCUU 1612 D-11051094-1112 977-995 GGCUUCAAGAACUGGAGGCUU 110 GCCUCCAGUUCUUGAAGCCUU 1613D-1106 1095-1113 978-996 GCUUCAAGAACUGGAGGCCUU 111 GGCCUCCAGUUCUUGAAGCUU1614 D-1107 1096-1114 979-997 CUUCAAGAACUGGAGGCCGUU 112CGGCCUCCAGUUCUUGAAGUU 1615 D-1108 1097-1115 980-998UUCAAGAACUGGAGGCCGGUU 113 CCGGCCUCCAGUUCUUGAAUU 1616 D-1109 1098-1116981-999 UCAAGAACUGGAGGCCGGAUU 114 UCCGGCCUCCAGUUCUUGAUU 1617 D-11101099-1117  982-1000 CAAGAACUGGAGGCCGGAGUU 115 CUCCGGCCUCCAGUUCUUGUU 1618D-1111 1100-1118  983-1001 AAGAACUGGAGGCCGGAGCUU 116GCUCCGGCCUCCAGUUCUUUU 1619 D-1112 1101-1119  984-1002AGAACUGGAGGCCGGAGCAUU 117 UGCUCCGGCCUCCAGUUCUUU 1620 D-1113 110-128110-128 CUCCCCUAAGUUCCAAUCCUU 118 GGAUUGGAACUUAGGGGAGUU 1621 D-11141102-1120  985-1003 GAACUGGAGGCCGGAGCAGUU 119 CUGCUCCGGCCUCCAGUUCUU 1622D-1115 1103-1121  986-1004 AACUGGAGGCCGGAGCAGCUU 120GCUGCUCCGGCCUCCAGUUUU 1623 D-1116 1104-1122  987-1005ACUGGAGGCCGGAGCAGCCUU 121 GGCUGCUCCGGCCUCCAGUUU 1624 D-1117 1105-1123 988-1006 CUGGAGGCCGGAGCAGCCGUU 122 CGGCUGCUCCGGCCUCCAGUU 1625 D-11181106-1124  989-1007 UGGAGGCCGGAGCAGCCGGUU 123 CCGGCUGCUCCGGCCUCCAUU 1626D-1119 1107-1125  990-1008 GGAGGCCGGAGCAGCCGGAUU 124UCCGGCUGCUCCGGCCUCCUU 1627 D-1120 1108-1126  991-1009GAGGCCGGAGCAGCCGGACUU 125 GUCCGGCUGCUCCGGCCUCUU 1628 D-1121 1109-1127 992-1010 AGGCCGGAGCAGCCGGACGUU 126 CGUCCGGCUGCUCCGGCCUUU 1629 D-11221110-1128  993-1011 GGCCGGAGCAGCCGGACGAUU 127 UCGUCCGGCUGCUCCGGCCUU 1630D-1123 1111-1129  994-1012 GCCGGAGCAGCCGGACGACUU 128GUCGUCCGGCUGCUCCGGCUU 1631 D-1124 111-129 111-129 UCCCCUAAGUUCCAAUCCAUU129 UGGAUUGGAACUUAGGGGAUU 1632 D-1125 1112-1130  995-1013CCGGAGCAGCCGGACGACUUU 130 AGUCGUCCGGCUGCUCCGGUU 1633 D-1126 1113-1131 996-1014 CGGAGCAGCCGGACGACUGUU 131 CAGUCGUCCGGCUGCUCCGUU 1634 D-11271114-1132  997-1015 GGAGCAGCCGGACGACUGGUU 132 CCAGUCGUCCGGCUGCUCCUU 1635D-1128 1115-1133  998-1016 GAGCAGCCGGACGACUGGUUU 133ACCAGUCGUCCGGCUGCUCUU 1636 D-1129 1116-1134  999-1017AGCAGCCGGACGACUGGUAUU 134 UACCAGUCGUCCGGCUGCUUU 1637 D-1130 1117-11351000-1018 GCAGCCGGACGACUGGUACUU 135 GUACCAGUCGUCCGGCUGCUU 1638 D-11311118-1136 1001-1019 CAGCCGGACGACUGGUACGUU 136 CGUACCAGUCGUCCGGCUGUU 1639D-1132 1119-1137 1002-1020 AGCCGGACGACUGGUACGGUU 137CCGUACCAGUCGUCCGGCUUU 1640 D-1133 1120-1138 1003-1021GCCGGACGACUGGUACGGCUU 138 GCCGUACCAGUCGUCCGGCUU 1641 D-1134 1121-11391004-1022 CCGGACGACUGGUACGGCCUU 139 GGCCGUACCAGUCGUCCGGUU 1642 D-1135112-130 112-130 CCCCUAAGUUCCAAUCCAUUU 140 AUGGAUUGGAACUUAGGGGUU 1643D-1136 1122-1140 1005-1023 CGGACGACUGGUACGGCCAUU 141UGGCCGUACCAGUCGUCCGUU 1644 D-1137 1123-1141 1006-1024GGACGACUGGUACGGCCACUU 142 GUGGCCGUACCAGUCGUCCUU 1645 D-1138 1124-11421007-1025 GACGACUGGUACGGCCACGUU 143 CGUGGCCGUACCAGUCGUCUU 1646 D-11391125-1143 1008-1026 ACGACUGGUACGGCCACGGUU 144 CCGUGGCCGUACCAGUCGUUU 1647D-1140 1126-1144 1009-1027 CGACUGGUACGGCCACGGGUU 145CCCGUGGCCGUACCAGUCGUU 1648 D-1141 1127-1145 1010-1028GACUGGUACGGCCACGGGCUU 146 GCCCGUGGCCGUACCAGUCUU 1649 D-1142 1128-11461011-1029 ACUGGUACGGCCACGGGCUUU 147 AGCCCGUGGCCGUACCAGUUU 1650 D-114311-29 11-29 GCACGGAAGAGUGAGGUGAUU 148 UCACCUCACUCUUCCGUGCUU 1651 D-11441129-1147 1012-1030 CUGGUACGGCCACGGGCUCUU 149 GAGCCCGUGGCCGUACCAGUU 1652D-1145 1130-1148 1013-1031 UGGUACGGCCACGGGCUCGUU 150CGAGCCCGUGGCCGUACCAUU 1653 D-1146 1131-1149 1014-1032GGUACGGCCACGGGCUCGGUU 151 CCGAGCCCGUGGCCGUACCUU 1654 D-1147 113-131113-131 CCCUAAGUUCCAAUCCAUUUU 152 AAUGGAUUGGAACUUAGGGUU 1655 D-11481132-1150 1015-1033 GUACGGCCACGGGCUCGGAUU 153 UCCGAGCCCGUGGCCGUACUU 1656D-1149 1133-1151 1016-1034 UACGGCCACGGGCUCGGAGUU 154CUCCGAGCCCGUGGCCGUAUU 1657 D-1150 1134-1152 1017-1035ACGGCCACGGGCUCGGAGGUU 155 CCUCCGAGCCCGUGGCCGUUU 1658 D-1151 1135-11531018-1036 CGGCCACGGGCUCGGAGGAUU 156 UCCUCCGAGCCCGUGGCCGUU 1659 D-11521136-1154 1019-1037 GGCCACGGGCUCGGAGGAGUU 157 CUCCUCCGAGCCCGUGGCCUU 1660D-1153 1137-1155 1020-1038 GCCACGGGCUCGGAGGAGGUU 158CCUCCUCCGAGCCCGUGGCUU 1661 D-1154 1138-1156 1021-1039CCACGGGCUCGGAGGAGGCUU 159 GCCUCCUCCGAGCCCGUGGUU 1662 D-1155 1139-11571022-1040 CACGGGCUCGGAGGAGGCGUU 160 CGCCUCCUCCGAGCCCGUGUU 1663 D-11561140-1158 1023-1041 ACGGGCUCGGAGGAGGCGAUU 161 UCGCCUCCUCCGAGCCCGUUU 1664D-1157 1141-1159 1024-1042 CGGGCUCGGAGGAGGCGAGUU 162CUCGCCUCCUCCGAGCCCGUU 1665 D-1158 114-132 114-132 CCUAAGUUCCAAUCCAUUUUU163 AAAUGGAUUGGAACUUAGGUU 1666 D-1159 1142-1160 1025-1043GGGCUCGGAGGAGGCGAGGUU 164 CCUCGCCUCCUCCGAGCCCUU 1667 D-1160 1143-11611026-1044 GGCUCGGAGGAGGCGAGGAUU 165 UCCUCGCCUCCUCCGAGCCUU 1668 D-11611144-1162 1027-1045 GCUCGGAGGAGGCGAGGACUU 166 GUCCUCGCCUCCUCCGAGCUU 1669D-1162 1145-1163 1028-1046 CUCGGAGGAGGCGAGGACUUU 167AGUCCUCGCCUCCUCCGAGUU 1670 D-1163 1146-1164 1029-1047UCGGAGGAGGCGAGGACUGUU 168 CAGUCCUCGCCUCCUCCGAUU 1671 D-1164 1147-11651030-1048 CGGAGGAGGCGAGGACUGUUU 169 ACAGUCCUCGCCUCCUCCGUU 1672 D-11651148-1166 1031-1049 GGAGGAGGCGAGGACUGUGUU 170 CACAGUCCUCGCCUCCUCCUU 1673D-1166 1149-1167 1032-1050 GAGGAGGCGAGGACUGUGCUU 171GCACAGUCCUCGCCUCCUCUU 1674 D-1167 1150-1168 1033-1051AGGAGGCGAGGACUGUGCCUU 172 GGCACAGUCCUCGCCUCCUUU 1675 D-1168 1151-11691034-1052 GGAGGCGAGGACUGUGCCCUU 173 GGGCACAGUCCUCGCCUCCUU 1676 D-1169115-133 115-133 CUAAGUUCCAAUCCAUUUCUU 174 GAAAUGGAUUGGAACUUAGUU 1677D-1170 1152-1170 1035-1053 GAGGCGAGGACUGUGCCCAUU 175UGGGCACAGUCCUCGCCUCUU 1678 D-1171 1153-1171 1036-1054AGGCGAGGACUGUGCCCACUU 176 GUGGGCACAGUCCUCGCCUUU 1679 D-1172 1154-11721037-1055 GGCGAGGACUGUGCCCACUUU 177 AGUGGGCACAGUCCUCGCCUU 1680 D-11731155-1173 1038-1056 GCGAGGACUGUGCCCACUUUU 178 AAGUGGGCACAGUCCUCGCUU 1681D-1174 1156-1174 1039-1057 CGAGGACUGUGCCCACUUCUU 179GAAGUGGGCACAGUCCUCGUU 1682 D-1175 1157-1175 1040-1058GAGGACUGUGCCCACUUCAUU 180 UGAAGUGGGCACAGUCCUCUU 1683 D-1176 1158-11761041-1059 AGGACUGUGCCCACUUCACUU 181 GUGAAGUGGGCACAGUCCUUU 1684 D-11771159-1177 1042-1060 GGACUGUGCCCACUUCACCUU 182 GGUGAAGUGGGCACAGUCCUU 1685D-1178 1160-1178 1043-1061 GACUGUGCCCACUUCACCGUU 183CGGUGAAGUGGGCACAGUCUU 1686 D-1179 1161-1179 1044-1062ACUGUGCCCACUUCACCGAUU 184 UCGGUGAAGUGGGCACAGUUU 1687 D-1180 116-134116-134 UAAGUUCCAAUCCAUUUCCUU 185 GGAAAUGGAUUGGAACUUAUU 1688 D-11811162-1180 1045-1063 CUGUGCCCACUUCACCGACUU 186 GUCGGUGAAGUGGGCACAGUU 1689D-1182 1163-1181 1046-1064 UGUGCCCACUUCACCGACGUU 187CGUCGGUGAAGUGGGCACAUU 1690 D-1183 1164-1182 1047-1065GUGCCCACUUCACCGACGAUU 188 UCGUCGGUGAAGUGGGCACUU 1691 D-1184 1165-11831048-1066 UGCCCACUUCACCGACGACUU 189 GUCGUCGGUGAAGUGGGCAUU 1692 D-11851166-1184 1049-1067 GCCCACUUCACCGACGACGUU 190 CGUCGUCGGUGAAGUGGGCUU 1693D-1186 1167-1185 1050-1068 CCCACUUCACCGACGACGGUU 191CCGUCGUCGGUGAAGUGGGUU 1694 D-1187 1168-1186 1051-1069CCACUUCACCGACGACGGCUU 192 GCCGUCGUCGGUGAAGUGGUU 1695 D-1188 1169-11871052-1070 CACUUCACCGACGACGGCCUU 193 GGCCGUCGUCGGUGAAGUGUU 1696 D-11891170-1188 1053-1071 ACUUCACCGACGACGGCCGUU 194 CGGCCGUCGUCGGUGAAGUUU 1697D-1190 1171-1189 1054-1072 CUUCACCGACGACGGCCGCUU 195GCGGCCGUCGUCGGUGAAGUU 1698 D-1191 117-135 117-135 AAGUUCCAAUCCAUUUCCAUU196 UGGAAAUGGAUUGGAACUUUU 1699 D-1192 1172-1190 1055-1073UUCACCGACGACGGCCGCUUU 197 AGCGGCCGUCGUCGGUGAAUU 1700 D-1193 1173-11911056-1074 UCACCGACGACGGCCGCUGUU 198 CAGCGGCCGUCGUCGGUGAUU 1701 D-11941174-1192 1057-1075 CACCGACGACGGCCGCUGGUU 199 CCAGCGGCCGUCGUCGGUGUU 1702D-1195 1175-1193 1058-1076 ACCGACGACGGCCGCUGGAUU 200UCCAGCGGCCGUCGUCGGUUU 1703 D-1196 1176-1194 1059-1077CCGACGACGGCCGCUGGAAUU 201 UUCCAGCGGCCGUCGUCGGUU 1704 D-1197 1177-11951060-1078 CGACGACGGCCGCUGGAACUU 202 GUUCCAGCGGCCGUCGUCGUU 1705 D-11981178-1196 1061-1079 GACGACGGCCGCUGGAACGUU 203 CGUUCCAGCGGCCGUCGUCUU 1706D-1199 1179-1197 1062-1080 ACGACGGCCGCUGGAACGAUU 204UCGUUCCAGCGGCCGUCGUUU 1707 D-1200 1180-1198 1063-1081CGACGGCCGCUGGAACGACUU 205 GUCGUUCCAGCGGCCGUCGUU 1708 D-1201 1181-11991064-1082 GACGGCCGCUGGAACGACGUU 206 CGUCGUUCCAGCGGCCGUCUU 1709 D-1202118-136 118-136 AGUUCCAAUCCAUUUCCACUU 207 GUGGAAAUGGAUUGGAACUUU 1710D-1203 1182-1200 1065-1083 ACGGCCGCUGGAACGACGAUU 208UCGUCGUUCCAGCGGCCGUUU 1711 D-1204 1183-1201 1066-1084CGGCCGCUGGAACGACGACUU 209 GUCGUCGUUCCAGCGGCCGUU 1712 D-1205 1184-12021067-1085 GGCCGCUGGAACGACGACGUU 210 CGUCGUCGUUCCAGCGGCCUU 1713 D-12061185-1203 1068-1086 GCCGCUGGAACGACGACGUUU 211 ACGUCGUCGUUCCAGCGGCUU 1714D-1207 1186-1204 1069-1087 CCGCUGGAACGACGACGUCUU 212GACGUCGUCGUUCCAGCGGUU 1715 D-1208 1187-1205 1070-1088CGCUGGAACGACGACGUCUUU 213 AGACGUCGUCGUUCCAGCGUU 1716 D-1209 1188-12061071-1089 GCUGGAACGACGACGUCUGUU 214 CAGACGUCGUCGUUCCAGCUU 1717 D-12101189-1207 1072-1090 CUGGAACGACGACGUCUGCUU 215 GCAGACGUCGUCGUUCCAGUU 1718D-1211  1-19  1-19 CCCAAACGGUGCACGGAAGUU 216 CUUCCGUGCACCGUUUGGGUU 1719D-1212 1190-1208 1073-1091 UGGAACGACGACGUCUGCCUU 217GGCAGACGUCGUCGUUCCAUU 1720 D-1213 1191-1209 1074-1092GGAACGACGACGUCUGCCAUU 218 UGGCAGACGUCGUCGUUCCUU 1721 D-1214 119-137119-137 GUUCCAAUCCAUUUCCACCUU 219 GGUGGAAAUGGAUUGGAACUU 1722 D-12151192-1210 1075-1093 GAACGACGACGUCUGCCAGUU 220 CUGGCAGACGUCGUCGUUCUU 1723D-1216 1193-1211 1076-1094 AACGACGACGUCUGCCAGAUU 221UCUGGCAGACGUCGUCGUUUU 1724 D-1217 1194-1212 1077-1095ACGACGACGUCUGCCAGAGUU 222 CUCUGGCAGACGUCGUCGUUU 1725 D-1218 1195-12131078-1096 CGACGACGUCUGCCAGAGGUU 223 CCUCUGGCAGACGUCGUCGUU 1726 D-12191196-1214 1079-1097 GACGACGUCUGCCAGAGGCUU 224 GCCUCUGGCAGACGUCGUCUU 1727D-1220 1197-1215 1080-1098 ACGACGUCUGCCAGAGGCCUU 225GGCCUCUGGCAGACGUCGUUU 1728 D-1221 1198-1216 1081-1099CGACGUCUGCCAGAGGCCCUU 226 GGGCCUCUGGCAGACGUCGUU 1729 D-1222 1199-12171082-1100 GACGUCUGCCAGAGGCCCUUU 227 AGGGCCUCUGGCAGACGUCUU 1730 D-12231200-1218 1083-1101 ACGUCUGCCAGAGGCCCUAUU 228 UAGGGCCUCUGGCAGACGUUU 1731D-1224 1201-1219 1084-1102 CGUCUGCCAGAGGCCCUACUU 229GUAGGGCCUCUGGCAGACGUU 1732 D-1225 120-138 120-138 UUCCAAUCCAUUUCCACCUUU230 AGGUGGAAAUGGAUUGGAAUU 1733 D-1226 1202-1220 1085-1103GUCUGCCAGAGGCCCUACCUU 231 GGUAGGGCCUCUGGCAGACUU 1734 D-1227 1203-12211086-1104 UCUGCCAGAGGCCCUACCGUU 232 CGGUAGGGCCUCUGGCAGAUU 1735 D-12281204-1222 1087-1105 CUGCCAGAGGCCCUACCGCUU 233 GCGGUAGGGCCUCUGGCAGUU 1736D-1229 1205-1223 1088-1106 UGCCAGAGGCCCUACCGCUUU 234AGCGGUAGGGCCUCUGGCAUU 1737 D-1230 1206-1224 1089-1107GCCAGAGGCCCUACCGCUGUU 235 CAGCGGUAGGGCCUCUGGCUU 1738 D-1231 1207-12251090-1108 CCAGAGGCCCUACCGCUGGUU 236 CCAGCGGUAGGGCCUCUGGUU 1739 D-12321208-1226 1091-1109 CAGAGGCCCUACCGCUGGGUU 237 CCCAGCGGUAGGGCCUCUGUU 1740D-1233 1209-1227 1092-1110 AGAGGCCCUACCGCUGGGUUU 238ACCCAGCGGUAGGGCCUCUUU 1741 D-1234 1210-1228 1093-1111GAGGCCCUACCGCUGGGUCUU 239 GACCCAGCGGUAGGGCCUCUU 1742 D-1235 1211-12291094-1112 AGGCCCUACCGCUGGGUCUUU 240 AGACCCAGCGGUAGGGCCUUU 1743 D-1236121-139 121-139 UCCAAUCCAUUUCCACCUCUU 241 GAGGUGGAAAUGGAUUGGAUU 1744D-1237 1212-1230 1095-1113 GGCCCUACCGCUGGGUCUGUU 242CAGACCCAGCGGUAGGGCCUU 1745 D-1238 1213-1231 1096-1114GCCCUACCGCUGGGUCUGCUU 243 GCAGACCCAGCGGUAGGGCUU 1746 D-1239 1214-12321097-1115 CCCUACCGCUGGGUCUGCGUU 244 CGCAGACCCAGCGGUAGGGUU 1747 D-12401215-1233 1098-1116 CCUACCGCUGGGUCUGCGAUU 245 UCGCAGACCCAGCGGUAGGUU 1748D-1241 1216-1234 1099-1117 CUACCGCUGGGUCUGCGAGUU 246CUCGCAGACCCAGCGGUAGUU 1749 D-1242 1217-1235 1100-1118UACCGCUGGGUCUGCGAGAUU 247 UCUCGCAGACCCAGCGGUAUU 1750 D-1243 1218-12361101-1119 ACCGCUGGGUCUGCGAGACUU 248 GUCUCGCAGACCCAGCGGUUU 1751 D-12441219-1237 1102-1120 CCGCUGGGUCUGCGAGACAUU 249 UGUCUCGCAGACCCAGCGGUU 1752D-1245 1220-1238 1103-1121 CGCUGGGUCUGCGAGACAGUU 250CUGUCUCGCAGACCCAGCGUU 1753 D-1246 1221-1239 1104-1122GCUGGGUCUGCGAGACAGAUU 251 UCUGUCUCGCAGACCCAGCUU 1754 D-1247 122-140122-140 CCAAUCCAUUUCCACCUCUUU 252 AGAGGUGGAAAUGGAUUGGUU 1755 D-12481222-1240 1105-1123 CUGGGUCUGCGAGACAGAGUU 253 CUCUGUCUCGCAGACCCAGUU 1756D-1249 1223-1241 1106-1124 UGGGUCUGCGAGACAGAGCUU 254GCUCUGUCUCGCAGACCCAUU 1757 D-1250 1224-1242 1107-1125GGGUCUGCGAGACAGAGCUUU 255 AGCUCUGUCUCGCAGACCCUU 1758 D-1251 1225-12431108-1126 GGUCUGCGAGACAGAGCUGUU 256 CAGCUCUGUCUCGCAGACCUU 1759 D-12521226-1244 1109-1127 GUCUGCGAGACAGAGCUGGUU 257 CCAGCUCUGUCUCGCAGACUU 1760D-1253 1227-1245 1110-1128 UCUGCGAGACAGAGCUGGAUU 258UCCAGCUCUGUCUCGCAGAUU 1761 D-1254 1228-1246 1111-1129CUGCGAGACAGAGCUGGACUU 259 GUCCAGCUCUGUCUCGCAGUU 1762 D-1255 1229-12471112-1130 UGCGAGACAGAGCUGGACAUU 260 UGUCCAGCUCUGUCUCGCAUU 1763 D-125612-30 12-30 CACGGAAGAGUGAGGUGACUU 261 GUCACCUCACUCUUCCGUGUU 1764 D-12571230-1248 1113-1131 GCGAGACAGAGCUGGACAAUU 262 UUGUCCAGCUCUGUCUCGCUU 1765D-1258 1231-1249 1114-1132 CGAGACAGAGCUGGACAAGUU 263CUUGUCCAGCUCUGUCUCGUU 1766 D-1259 123-141 123-141 CAAUCCAUUUCCACCUCUGUU264 CAGAGGUGGAAAUGGAUUGUU 1767 D-1260 1232-1250 1115-1133GAGACAGAGCUGGACAAGGUU 265 CCUUGUCCAGCUCUGUCUCUU 1768 D-1261 1233-12511116-1134 AGACAGAGCUGGACAAGGCUU 266 GCCUUGUCCAGCUCUGUCUUU 1769 D-12621234-1252 1117-1135 GACAGAGCUGGACAAGGCCUU 267 GGCCUUGUCCAGCUCUGUCUU 1770D-1263 1235-1253 1118-1136 ACAGAGCUGGACAAGGCCAUU 268UGGCCUUGUCCAGCUCUGUUU 1771 D-1264 1236-1254 1119-1137CAGAGCUGGACAAGGCCAGUU 269 CUGGCCUUGUCCAGCUCUGUU 1772 D-1265 1237-12551120-1138 AGAGCUGGACAAGGCCAGCUU 270 GCUGGCCUUGUCCAGCUCUUU 1773 D-12661238-1256 1121-1139 GAGCUGGACAAGGCCAGCCUU 271 GGCUGGCCUUGUCCAGCUCUU 1774D-1267 1239-1257 1122-1140 AGCUGGACAAGGCCAGCCAUU 272UGGCUGGCCUUGUCCAGCUUU 1775 D-1268 1240-1258 1123-1141GCUGGACAAGGCCAGCCAGUU 273 CUGGCUGGCCUUGUCCAGCUU 1776 D-1269 1241-12591124-1142 CUGGACAAGGCCAGCCAGGUU 274 CCUGGCUGGCCUUGUCCAGUU 1777 D-1270124-142 124-142 AAUCCAUUUCCACCUCUGUUU 275 ACAGAGGUGGAAAUGGAUUUU 1778D-1271 1242-1260 1125-1143 UGGACAAGGCCAGCCAGGAUU 276UCCUGGCUGGCCUUGUCCAUU 1779 D-1272 1243-1261 1126-1144GGACAAGGCCAGCCAGGAGUU 277 CUCCUGGCUGGCCUUGUCCUU 1780 D-1273 1244-12621127-1145 GACAAGGCCAGCCAGGAGCUU 278 GCUCCUGGCUGGCCUUGUCUU 1781 D-12741245-1263 1128-1146 ACAAGGCCAGCCAGGAGCCUU 279 GGCUCCUGGCUGGCCUUGUUU 1782D-1275 1246-1264 1129-1147 CAAGGCCAGCCAGGAGCCAUU 280UGGCUCCUGGCUGGCCUUGUU 1783 D-1276 1247-1265 1130-1148AAGGCCAGCCAGGAGCCACUU 281 GUGGCUCCUGGCUGGCCUUUU 1784 D-1277 1248-12661131-1149 AGGCCAGCCAGGAGCCACCUU 282 GGUGGCUCCUGGCUGGCCUUU 1785 D-12781249-1267 1132-1150 GGCCAGCCAGGAGCCACCUUU 283 AGGUGGCUCCUGGCUGGCCUU 1786D-1279 1250-1268 1133-1151 GCCAGCCAGGAGCCACCUCUU 284GAGGUGGCUCCUGGCUGGCUU 1787 D-1280 1251-1269 1134-1152CCAGCCAGGAGCCACCUCUUU 285 AGAGGUGGCUCCUGGCUGGUU 1788 D-1281 125-143125-143 AUCCAUUUCCACCUCUGUUUU 286 AACAGAGGUGGAAAUGGAUUU 1789 D-12821252-1270 1135-1153 CAGCCAGGAGCCACCUCUCUU 287 GAGAGGUGGCUCCUGGCUGUU 1790D-1283 1253-1271 1136-1154 AGCCAGGAGCCACCUCUCCUU 288GGAGAGGUGGCUCCUGGCUUU 1791 D-1284 1254-1272 1137-1155GCCAGGAGCCACCUCUCCUUU 289 AGGAGAGGUGGCUCCUGGCUU 1792 D-1285 1255-12731138-1156 CCAGGAGCCACCUCUCCUUUU 290 AAGGAGAGGUGGCUCCUGGUU 1793 D-12861256-1274 1139-1157 CAGGAGCCACCUCUCCUUUUU 291 AAAGGAGAGGUGGCUCCUGUU 1794D-1287 1257-1275 1140-1158 AGGAGCCACCUCUCCUUUAUU 292UAAAGGAGAGGUGGCUCCUUU 1795 D-1288 1258-1276 1141-1159GGAGCCACCUCUCCUUUAAUU 293 UUAAAGGAGAGGUGGCUCCUU 1796 D-1289 1259-12771142-1160 GAGCCACCUCUCCUUUAAUUU 294 AUUAAAGGAGAGGUGGCUCUU 1797 D-12901260-1278 1143-1161 AGCCACCUCUCCUUUAAUUUU 295 AAUUAAAGGAGAGGUGGCUUU 1798D-1291 1261-1279 1144-1162 GCCACCUCUCCUUUAAUUUUU 296AAAUUAAAGGAGAGGUGGCUU 1799 D-1292 126-144 126-144 UCCAUUUCCACCUCUGUUUUU297 AAACAGAGGUGGAAAUGGAUU 1800 D-1293 1262-1280 1145-1163CCACCUCUCCUUUAAUUUAUU 298 UAAAUUAAAGGAGAGGUGGUU 1801 D-1294 1263-12811146-1164 CACCUCUCCUUUAAUUUAUUU 299 AUAAAUUAAAGGAGAGGUGUU 1802 D-12951264-1282 1147-1165 ACCUCUCCUUUAAUUUAUUUU 300 AAUAAAUUAAAGGAGAGGUUU 1803D-1296 1265-1283 1148-1166 CCUCUCCUUUAAUUUAUUUUU 301AAAUAAAUUAAAGGAGAGGUU 1804 D-1297 1266-1284 1149-1167CUCUCCUUUAAUUUAUUUCUU 302 GAAAUAAAUUAAAGGAGAGUU 1805 D-1298 1267-12851150-1168 UCUCCUUUAAUUUAUUUCUUU 303 AGAAAUAAAUUAAAGGAGAUU 1806 D-12991268-1286 1151-1169 CUCCUUUAAUUUAUUUCUUUU 304 AAGAAAUAAAUUAAAGGAGUU 1807D-1300 1269-1287 1152-1170 UCCUUUAAUUUAUUUCUUCUU 305GAAGAAAUAAAUUAAAGGAUU 1808 D-1301 1270-1288 1153-1171CCUUUAAUUUAUUUCUUCAUU 306 UGAAGAAAUAAAUUAAAGGUU 1809 D-1302 1271-12891154-1172 CUUUAAUUUAUUUCUUCAAUU 307 UUGAAGAAAUAAAUUAAAGUU 1810 D-1303127-145 127-145 CCAUUUCCACCUCUGUUUAUU 308 UAAACAGAGGUGGAAAUGGUU 1811D-1304 1272-1290 1155-1173 UUUAAUUUAUUUCUUCAAUUU 309AUUGAAGAAAUAAAUUAAAUU 1812 D-1305 1273-1291 1156-1174UUAAUUUAUUUCUUCAAUGUU 310 CAUUGAAGAAAUAAAUUAAUU 1813 D-1306 1274-12921157-1175 UAAUUUAUUUCUUCAAUGCUU 311 GCAUUGAAGAAAUAAAUUAUU 1814 D-13071275-1293 1158-1176 AAUUUAUUUCUUCAAUGCCUU 312 GGCAUUGAAGAAAUAAAUUUU 1815D-1308 1276-1294 1159-1177 AUUUAUUUCUUCAAUGCCUUU 313AGGCAUUGAAGAAAUAAAUUU 1816 D-1309 1277-1295 1160-1178UUUAUUUCUUCAAUGCCUCUU 314 GAGGCAUUGAAGAAAUAAAUU 1817 D-1310 1278-12961161-1179 UUAUUUCUUCAAUGCCUCGUU 315 CGAGGCAUUGAAGAAAUAAUU 1818 D-13111279-1297 1162-1180 UAUUUCUUCAAUGCCUCGAUU 316 UCGAGGCAUUGAAGAAAUAUU 1819D-1312 1280-1298 1163-1181 AUUUCUUCAAUGCCUCGACUU 317GUCGAGGCAUUGAAGAAAUUU 1820 D-1313 1281-1299 1164-1182UUUCUUCAAUGCCUCGACCUU 318 GGUCGAGGCAUUGAAGAAAUU 1821 D-1314 128-146128-146 CAUUUCCACCUCUGUUUACUU 319 GUAAACAGAGGUGGAAAUGUU 1822 D-13151282-1300 1165-1183 UUCUUCAAUGCCUCGACCUUU 320 AGGUCGAGGCAUUGAAGAAUU 1823D-1316 1283-1301 1166-1184 UCUUCAAUGCCUCGACCUGUU 321CAGGUCGAGGCAUUGAAGAUU 1824 D-1317 1284-1302 1167-1185CUUCAAUGCCUCGACCUGCUU 322 GCAGGUCGAGGCAUUGAAGUU 1825 D-1318 1285-13031168-1186 UUCAAUGCCUCGACCUGCCUU 323 GGCAGGUCGAGGCAUUGAAUU 1826 D-13191286-1304 1169-1187 UCAAUGCCUCGACCUGCCGUU 324 CGGCAGGUCGAGGCAUUGAUU 1827D-1320 1287-1305 1170-1188 CAAUGCCUCGACCUGCCGCUU 325GCGGCAGGUCGAGGCAUUGUU 1828 D-1321 1288-1306 1171-1189AAUGCCUCGACCUGCCGCAUU 326 UGCGGCAGGUCGAGGCAUUUU 1829 D-1322 1289-13071172-1190 AUGCCUCGACCUGCCGCAGUU 327 CUGCGGCAGGUCGAGGCAUUU 1830 D-13231290-1308 1173-1191 UGCCUCGACCUGCCGCAGGUU 328 CCUGCGGCAGGUCGAGGCAUU 1831D-1324 1291-1309 1174-1192 GCCUCGACCUGCCGCAGGGUU 329CCCUGCGGCAGGUCGAGGCUU 1832 D-1325 129-147 129-147 AUUUCCACCUCUGUUUACUUU330 AGUAAACAGAGGUGGAAAUUU 1833 D-1326 1292-1310 1175-1193CCUCGACCUGCCGCAGGGGUU 331 CCCCUGCGGCAGGUCGAGGUU 1834 D-1327 1293-13111176-1194 CUCGACCUGCCGCAGGGGUUU 332 ACCCCUGCGGCAGGUCGAGUU 1835 D-13281294-1312 1177-1195 UCGACCUGCCGCAGGGGUCUU 333 GACCCCUGCGGCAGGUCGAUU 1836D-1329 1295-1313 1178-1196 CGACCUGCCGCAGGGGUCCUU 334GGACCCCUGCGGCAGGUCGUU 1837 D-1330 1296-1314 1179-1197GACCUGCCGCAGGGGUCCGUU 335 CGGACCCCUGCGGCAGGUCUU 1838 D-1331 1297-13151180-1198 ACCUGCCGCAGGGGUCCGGUU 336 CCGGACCCCUGCGGCAGGUUU 1839 D-13321298-1316 1181-1199 CCUGCCGCAGGGGUCCGGGUU 337 CCCGGACCCCUGCGGCAGGUU 1840D-1333 1299-1317 1182-1200 CUGCCGCAGGGGUCCGGGAUU 338UCCCGGACCCCUGCGGCAGUU 1841 D-1334 1300-1318 1183-1201UGCCGCAGGGGUCCGGGAUUU 339 AUCCCGGACCCCUGCGGCAUU 1842 D-1335 1301-13191184-1202 GCCGCAGGGGUCCGGGAUUUU 340 AAUCCCGGACCCCUGCGGCUU 1843 D-1336130-148 130-148 UUUCCACCUCUGUUUACUGUU 341 CAGUAAACAGAGGUGGAAAUU 1844D-1337 1302-1320 1185-1203 CCGCAGGGGUCCGGGAUUGUU 342CAAUCCCGGACCCCUGCGGUU 1845 D-1338 1303-1321 1186-1204CGCAGGGGUCCGGGAUUGGUU 343 CCAAUCCCGGACCCCUGCGUU 1846 D-1339 1304-13221187-1205 GCAGGGGUCCGGGAUUGGGUU 344 CCCAAUCCCGGACCCCUGCUU 1847 D-13401305-1323 1188-1206 CAGGGGUCCGGGAUUGGGAUU 345 UCCCAAUCCCGGACCCCUGUU 1848D-1341 1306-1324 1189-1207 AGGGGUCCGGGAUUGGGAAUU 346UUCCCAAUCCCGGACCCCUUU 1849 D-1342 1307-1325 1190-1208GGGGUCCGGGAUUGGGAAUUU 347 AUUCCCAAUCCCGGACCCCUU 1850 D-1343 1308-13261191-1209 GGGUCCGGGAUUGGGAAUCUU 348 GAUUCCCAAUCCCGGACCCUU 1851 D-13441309-1327 1192-1210 GGUCCGGGAUUGGGAAUCCUU 349 GGAUUCCCAAUCCCGGACCUU 1852D-1345 1310-1328 1193-1211 GUCCGGGAUUGGGAAUCCGUU 350CGGAUUCCCAAUCCCGGACUU 1853 D-1346 1311-1329 1194-1212UCCGGGAUUGGGAAUCCGCUU 351 GCGGAUUCCCAAUCCCGGAUU 1854 D-1347 131-149131-149 UUCCACCUCUGUUUACUGUUU 352 ACAGUAAACAGAGGUGGAAUU 1855 D-13481312-1330 1195-1213 CCGGGAUUGGGAAUCCGCCUU 353 GGCGGAUUCCCAAUCCCGGUU 1856D-1349 1313-1331 1196-1214 CGGGAUUGGGAAUCCGCCCUU 354GGGCGGAUUCCCAAUCCCGUU 1857 D-1350 1314-1332 1197-1215GGGAUUGGGAAUCCGCCCAUU 355 UGGGCGGAUUCCCAAUCCCUU 1858 D-1351 1315-13331198-1216 GGAUUGGGAAUCCGCCCAUUU 356 AUGGGCGGAUUCCCAAUCCUU 1859 D-13521316-1334 1199-1217 GAUUGGGAAUCCGCCCAUCUU 357 GAUGGGCGGAUUCCCAAUCUU 1860D-1353 1317-1335 1200-1218 AUUGGGAAUCCGCCCAUCUUU 358AGAUGGGCGGAUUCCCAAUUU 1861 D-1354 1318-1336 1201-1219UUGGGAAUCCGCCCAUCUGUU 359 CAGAUGGGCGGAUUCCCAAUU 1862 D-1355 1319-13371202-1220 UGGGAAUCCGCCCAUCUGGUU 360 CCAGAUGGGCGGAUUCCCAUU 1863 D-13561320-1338 1203-1221 GGGAAUCCGCCCAUCUGGGUU 361 CCCAGAUGGGCGGAUUCCCUU 1864D-1357 1321-1339 1204-1222 GGAAUCCGCCCAUCUGGGGUU 362CCCCAGAUGGGCGGAUUCCUU 1865 D-1358 132-150 132-150 UCCACCUCUGUUUACUGUCUU363 GACAGUAAACAGAGGUGGAUU 1866 D-1359 1322-1340 1205-1223GAAUCCGCCCAUCUGGGGGUU 364 CCCCCAGAUGGGCGGAUUCUU 1867 D-1360 1323-13411206-1224 AAUCCGCCCAUCUGGGGGCUU 365 GCCCCCAGAUGGGCGGAUUUU 1868 D-13611324-1342 1207-1225 AUCCGCCCAUCUGGGGGCCUU 366 GGCCCCCAGAUGGGCGGAUUU 1869D-1362 1325-1343 1208-1226 UCCGCCCAUCUGGGGGCCUUU 367AGGCCCCCAGAUGGGCGGAUU 1870 D-1363 1326-1344 1209-1227CCGCCCAUCUGGGGGCCUCUU 368 GAGGCCCCCAGAUGGGCGGUU 1871 D-1364 1327-13451210-1228 CGCCCAUCUGGGGGCCUCUUU 369 AGAGGCCCCCAGAUGGGCGUU 1872 D-13651328-1346 1211-1229 GCCCAUCUGGGGGCCUCUUUU 370 AAGAGGCCCCCAGAUGGGCUU 1873D-1366 1329-1347 1212-1230 CCCAUCUGGGGGCCUCUUCUU 371GAAGAGGCCCCCAGAUGGGUU 1874 D-1367 1330-1348 1213-1231CCAUCUGGGGGCCUCUUCUUU 372 AGAAGAGGCCCCCAGAUGGUU 1875 D-1368 13-31 13-31ACGGAAGAGUGAGGUGACUUU 373 AGUCACCUCACUCUUCCGUUU 1876 D-1369 1331-13491214-1232 CAUCUGGGGGCCUCUUCUGUU 374 CAGAAGAGGCCCCCAGAUGUU 1877 D-1370133-151 133-151 CCACCUCUGUUUACUGUCCUU 375 GGACAGUAAACAGAGGUGGUU 1878D-1371 1332-1350 1215-1233 AUCUGGGGGCCUCUUCUGCUU 376GCAGAAGAGGCCCCCAGAUUU 1879 D-1372 1333-1351 1216-1234UCUGGGGGCCUCUUCUGCUUU 377 AGCAGAAGAGGCCCCCAGAUU 1880 D-1373 1334-13521217-1235 CUGGGGGCCUCUUCUGCUUUU 378 AAGCAGAAGAGGCCCCCAGUU 1881 D-13741335-1353 1218-1236 UGGGGGCCUCUUCUGCUUUUU 379 AAAGCAGAAGAGGCCCCCAUU 1882D-1375 1336-1354 1219-1237 GGGGGCCUCUUCUGCUUUCUU 380GAAAGCAGAAGAGGCCCCCUU 1883 D-1376 1337-1355 1220-1238GGGGCCUCUUCUGCUUUCUUU 381 AGAAAGCAGAAGAGGCCCCUU 1884 D-1377 1338-13561221-1239 GGGCCUCUUCUGCUUUCUCUU 382 GAGAAAGCAGAAGAGGCCCUU 1885 D-13781339-1357 1222-1240 GGCCUCUUCUGCUUUCUCGUU 383 CGAGAAAGCAGAAGAGGCCUU 1886D-1379 1340-1358 1223-1241 GCCUCUUCUGCUUUCUCGGUU 384CCGAGAAAGCAGAAGAGGCUU 1887 D-1380 1341-1359 1224-1242CCUCUUCUGCUUUCUCGGGUU 385 CCCGAGAAAGCAGAAGAGGUU 1888 D-1381 134-152134-152 CACCUCUGUUUACUGUCCAUU 386 UGGACAGUAAACAGAGGUGUU 1889 D-13821342-1360 1225-1243 CUCUUCUGCUUUCUCGGGAUU 387 UCCCGAGAAAGCAGAAGAGUU 1890D-1383 1343-1361 1226-1244 UCUUCUGCUUUCUCGGGAAUU 388UUCCCGAGAAAGCAGAAGAUU 1891 D-1384 1344-1362 1227-1245CUUCUGCUUUCUCGGGAAUUU 389 AUUCCCGAGAAAGCAGAAGUU 1892 D-1385 1345-13631228-1246 UUCUGCUUUCUCGGGAAUUUU 390 AAUUCCCGAGAAAGCAGAAUU 1893 D-13861346-1364 1229-1247 UCUGCUUUCUCGGGAAUUUUU 391 AAAUUCCCGAGAAAGCAGAUU 1894D-1387 1347-1365 1230-1248 CUGCUUUCUCGGGAAUUUUUU 392AAAAUUCCCGAGAAAGCAGUU 1895 D-1388 1348-1366 1231-1249UGCUUUCUCGGGAAUUUUCUU 393 GAAAAUUCCCGAGAAAGCAUU 1896 D-1389 1349-13671232-1250 GCUUUCUCGGGAAUUUUCAUU 394 UGAAAAUUCCCGAGAAAGCUU 1897 D-13901350-1368 1233-1251 CUUUCUCGGGAAUUUUCAUUU 395 AUGAAAAUUCCCGAGAAAGUU 1898D-1391 1351-1369 1234-1252 UUUCUCGGGAAUUUUCAUCUU 396GAUGAAAAUUCCCGAGAAAUU 1899 D-1392 135-153 135-153 ACCUCUGUUUACUGUCCAAUU397 UUGGACAGUAAACAGAGGUUU 1900 D-1393 1352-1370 1235-1253UUCUCGGGAAUUUUCAUCUUU 398 AGAUGAAAAUUCCCGAGAAUU 1901 D-1394 1353-13711236-1254 UCUCGGGAAUUUUCAUCUAUU 399 UAGAUGAAAAUUCCCGAGAUU 1902 D-13951354-1372 1237-1255 CUCGGGAAUUUUCAUCUAGUU 400 CUAGAUGAAAAUUCCCGAGUU 1903D-1396 1355-1373 1238-1256 UCGGGAAUUUUCAUCUAGGUU 401CCUAGAUGAAAAUUCCCGAUU 1904 D-1397 1356-1374 1239-1257CGGGAAUUUUCAUCUAGGAUU 402 UCCUAGAUGAAAAUUCCCGUU 1905 D-1398 1357-13751240-1258 GGGAAUUUUCAUCUAGGAUUU 403 AUCCUAGAUGAAAAUUCCCUU 1906 D-13991358-1376 1241-1259 GGAAUUUUCAUCUAGGAUUUU 404 AAUCCUAGAUGAAAAUUCCUU 1907D-1400 1359-1377 1242-1260 GAAUUUUCAUCUAGGAUUUUU 405AAAUCCUAGAUGAAAAUUCUU 1908 D-1401 1360-1378 1243-1261AAUUUUCAUCUAGGAUUUUUU 406 AAAAUCCUAGAUGAAAAUUUU 1909 D-1402 1361-13791244-1262 AUUUUCAUCUAGGAUUUUAUU 407 UAAAAUCCUAGAUGAAAAUUU 1910 D-1403136-154 136-154 CCUCUGUUUACUGUCCAAAUU 408 UUUGGACAGUAAACAGAGGUU 1911D-1404 1362-1380 1245-1263 UUUUCAUCUAGGAUUUUAAUU 409UUAAAAUCCUAGAUGAAAAUU 1912 D-1405 1363-1381 1246-1264UUUCAUCUAGGAUUUUAAGUU 410 CUUAAAAUCCUAGAUGAAAUU 1913 D-1406 1364-13821247-1265 UUCAUCUAGGAUUUUAAGGUU 411 CCUUAAAAUCCUAGAUGAAUU 1914 D-14071365-1383 1248-1266 UCAUCUAGGAUUUUAAGGGUU 412 CCCUUAAAAUCCUAGAUGAUU 1915D-1408 1366-1384 1249-1267 CAUCUAGGAUUUUAAGGGAUU 413UCCCUUAAAAUCCUAGAUGUU 1916 D-1409 1367-1385 1250-1268AUCUAGGAUUUUAAGGGAAUU 414 UUCCCUUAAAAUCCUAGAUUU 1917 D-1410 1368-13861251-1269 UCUAGGAUUUUAAGGGAAGUU 415 CUUCCCUUAAAAUCCUAGAUU 1918 D-14111369-1387 1252-1270 CUAGGAUUUUAAGGGAAGGUU 416 CCUUCCCUUAAAAUCCUAGUU 1919D-1412 1370-1388 1253-1271 UAGGAUUUUAAGGGAAGGGUU 417CCCUUCCCUUAAAAUCCUAUU 1920 D-1413 1371-1389 1254-1272AGGAUUUUAAGGGAAGGGGUU 418 CCCCUUCCCUUAAAAUCCUUU 1921 D-1414 137-155137-155 CUCUGUUUACUGUCCAAAGUU 419 CUUUGGACAGUAAACAGAGUU 1922 D-14151372-1390 1255-1273 GGAUUUUAAGGGAAGGGGAUU 420 UCCCCUUCCCUUAAAAUCCUU 1923D-1416 1373-1391 1256-1274 GAUUUUAAGGGAAGGGGAAUU 421UUCCCCUUCCCUUAAAAUCUU 1924 D-1417 1374-1392 1257-1275AUUUUAAGGGAAGGGGAAGUU 422 CUUCCCCUUCCCUUAAAAUUU 1925 D-1418 1375-13931258-1276 UUUUAAGGGAAGGGGAAGGUU 423 CCUUCCCCUUCCCUUAAAAUU 1926 D-14191376-1394 1259-1277 UUUAAGGGAAGGGGAAGGAUU 424 UCCUUCCCCUUCCCUUAAAUU 1927D-1420 1377-1395 1260-1278 UUAAGGGAAGGGGAAGGAUUU 425AUCCUUCCCCUUCCCUUAAUU 1928 D-1421 1378-1396 1261-1279UAAGGGAAGGGGAAGGAUAUU 426 UAUCCUUCCCCUUCCCUUAUU 1929 D-1422 1379-13971262-1280 AAGGGAAGGGGAAGGAUAGUU 427 CUAUCCUUCCCCUUCCCUUUU 1930 D-14231380-1398 1263-1281 AGGGAAGGGGAAGGAUAGGUU 428 CCUAUCCUUCCCCUUCCCUUU 1931D-1424 1381-1399 1264-1282 GGGAAGGGGAAGGAUAGGGUU 429CCCUAUCCUUCCCCUUCCCUU 1932 D-1425 138-156 138-156 UCUGUUUACUGUCCAAAGUUU430 ACUUUGGACAGUAAACAGAUU 1933 D-1426 1382-1400 1265-1283GGAAGGGGAAGGAUAGGGUUU 431 ACCCUAUCCUUCCCCUUCCUU 1934 D-1427 1383-14011266-1284 GAAGGGGAAGGAUAGGGUGUU 432 CACCCUAUCCUUCCCCUUCUU 1935 D-14281384-1402 1267-1285 AAGGGGAAGGAUAGGGUGAUU 433 UCACCCUAUCCUUCCCCUUUU 1936D-1429 1385-1403 1268-1286 AGGGGAAGGAUAGGGUGAUUU 434AUCACCCUAUCCUUCCCCUUU 1937 D-1430 1386-1404 1269-1287GGGGAAGGAUAGGGUGAUGUU 435 CAUCACCCUAUCCUUCCCCUU 1938 D-1431 1387-14051270-1288 GGGAAGGAUAGGGUGAUGUUU 436 ACAUCACCCUAUCCUUCCCUU 1939 D-14321388-1406 1271-1289 GGAAGGAUAGGGUGAUGUUUU 437 AACAUCACCCUAUCCUUCCUU 1940D-1433 1389-1407 1272-1290 GAAGGAUAGGGUGAUGUUCUU 438GAACAUCACCCUAUCCUUCUU 1941 D-1434 1390-1408 1273-1291AAGGAUAGGGUGAUGUUCCUU 439 GGAACAUCACCCUAUCCUUUU 1942 D-1435 1391-14091274-1292 AGGAUAGGGUGAUGUUCCGUU 440 CGGAACAUCACCCUAUCCUUU 1943 D-1436139-157 139-157 CUGUUUACUGUCCAAAGUCUU 441 GACUUUGGACAGUAAACAGUU 1944D-1437 1392-1410 1275-1293 GGAUAGGGUGAUGUUCCGAUU 442UCGGAACAUCACCCUAUCCUU 1945 D-1438 1393-1411 1276-1294GAUAGGGUGAUGUUCCGAAUU 443 UUCGGAACAUCACCCUAUCUU 1946 D-1439 1394-14121277-1295 AUAGGGUGAUGUUCCGAAGUU 444 CUUCGGAACAUCACCCUAUUU 1947 D-14401395-1413 1278-1296 UAGGGUGAUGUUCCGAAGGUU 445 CCUUCGGAACAUCACCCUAUU 1948D-1441 1396-1414 1279-1297 AGGGUGAUGUUCCGAAGGUUU 446ACCUUCGGAACAUCACCCUUU 1949 D-1442 1397-1415 1280-1298GGGUGAUGUUCCGAAGGUGUU 447 CACCUUCGGAACAUCACCCUU 1950 D-1443 1398-14161281-1299 GGUGAUGUUCCGAAGGUGAUU 448 UCACCUUCGGAACAUCACCUU 1951 D-14441399-1417 1282-1300 GUGAUGUUCCGAAGGUGAGUU 449 CUCACCUUCGGAACAUCACUU 1952D-1445 1400-1418 1283-1301 UGAUGUUCCGAAGGUGAGGUU 450CCUCACCUUCGGAACAUCAUU 1953 D-1446 1401-1419 1284-1302GAUGUUCCGAAGGUGAGGAUU 451 UCCUCACCUUCGGAACAUCUU 1954 D-1447 140-158140-158 UGUUUACUGUCCAAAGUCCUU 452 GGACUUUGGACAGUAAACAUU 1955 D-14481402-1420 1285-1303 AUGUUCCGAAGGUGAGGAGUU 453 CUCCUCACCUUCGGAACAUUU 1956D-1449 1403-1421 1286-1304 UGUUCCGAAGGUGAGGAGCUU 454GCUCCUCACCUUCGGAACAUU 1957 D-1450 1404-1422 1287-1305GUUCCGAAGGUGAGGAGCUUU 455 AGCUCCUCACCUUCGGAACUU 1958 D-1451 1405-14231288-1306 UUCCGAAGGUGAGGAGCUUUU 456 AAGCUCCUCACCUUCGGAAUU 1959 D-14521406-1424 1289-1307 UCCGAAGGUGAGGAGCUUGUU 457 CAAGCUCCUCACCUUCGGAUU 1960D-1453 1407-1425 1290-1308 CCGAAGGUGAGGAGCUUGAUU 458UCAAGCUCCUCACCUUCGGUU 1961 D-1454 1408-1426 1291-1309CGAAGGUGAGGAGCUUGAAUU 459 UUCAAGCUCCUCACCUUCGUU 1962 D-1455 1409-14271292-1310 GAAGGUGAGGAGCUUGAAAUU 460 UUUCAAGCUCCUCACCUUCUU 1963 D-14561410-1428 1293-1311 AAGGUGAGGAGCUUGAAACUU 461 GUUUCAAGCUCCUCACCUUUU 1964D-1457 1411-1429 1294-1312 AGGUGAGGAGCUUGAAACCUU 462GGUUUCAAGCUCCUCACCUUU 1965 D-1458 141-159 141-159 GUUUACUGUCCAAAGUCCCUU463 GGGACUUUGGACAGUAAACUU 1966 D-1459 1412-1430 1295-1313GGUGAGGAGCUUGAAACCCUU 464 GGGUUUCAAGCUCCUCACCUU 1967 D-1460 1413-14311296-1314 GUGAGGAGCUUGAAACCCGUU 465 CGGGUUUCAAGCUCCUCACUU 1968 D-14611414-1432 1297-1315 UGAGGAGCUUGAAACCCGUUU 466 ACGGGUUUCAAGCUCCUCAUU 1969D-1462 1415-1433 1298-1316 GAGGAGCUUGAAACCCGUGUU 467CACGGGUUUCAAGCUCCUCUU 1970 D-1463 1416-1434 1299-1317AGGAGCUUGAAACCCGUGGUU 468 CCACGGGUUUCAAGCUCCUUU 1971 D-1464 1417-14351300-1318 GGAGCUUGAAACCCGUGGCUU 469 GCCACGGGUUUCAAGCUCCUU 1972 D-14651418-1436 1301-1319 GAGCUUGAAACCCGUGGCGUU 470 CGCCACGGGUUUCAAGCUCUU 1973D-1466 1419-1437 1302-1320 AGCUUGAAACCCGUGGCGCUU 471GCGCCACGGGUUUCAAGCUUU 1974 D-1467 1420-1438 1303-1321GCUUGAAACCCGUGGCGCUUU 472 AGCGCCACGGGUUUCAAGCUU 1975 D-1468 1421-14391304-1322 CUUGAAACCCGUGGCGCUUUU 473 AAGCGCCACGGGUUUCAAGUU 1976 D-1469142-160 142-160 UUUACUGUCCAAAGUCCCGUU 474 CGGGACUUUGGACAGUAAAUU 1977D-1470 1422-1440 1305-1323 UUGAAACCCGUGGCGCUUUUU 475AAAGCGCCACGGGUUUCAAUU 1978 D-1471 1423-1441 1306-1324UGAAACCCGUGGCGCUUUCUU 476 GAAAGCGCCACGGGUUUCAUU 1979 D-1472 1424-14421307-1325 GAAACCCGUGGCGCUUUCUUU 477 AGAAAGCGCCACGGGUUUCUU 1980 D-14731425-1443 1308-1326 AAACCCGUGGCGCUUUCUGUU 478 CAGAAAGCGCCACGGGUUUUU 1981D-1474 1426-1444 1309-1327 AACCCGUGGCGCUUUCUGCUU 479GCAGAAAGCGCCACGGGUUUU 1982 D-1475 1427-1445 1310-1328ACCCGUGGCGCUUUCUGCAUU 480 UGCAGAAAGCGCCACGGGUUU 1983 D-1476 1428-14461311-1329 CCCGUGGCGCUUUCUGCAGUU 481 CUGCAGAAAGCGCCACGGGUU 1984 D-14771429-1447 1312-1330 CCGUGGCGCUUUCUGCAGUUU 482 ACUGCAGAAAGCGCCACGGUU 1985D-1478 1430-1448 1313-1331 CGUGGCGCUUUCUGCAGUUUU 483AACUGCAGAAAGCGCCACGUU 1986 D-1479 1431-1449 1314-1332GUGGCGCUUUCUGCAGUUUUU 484 AAACUGCAGAAAGCGCCACUU 1987 D-1480 143-161143-161 UUACUGUCCAAAGUCCCGGUU 485 CCGGGACUUUGGACAGUAAUU 1988 D-148114-32 14-32 CGGAAGAGUGAGGUGACUGUU 486 CAGUCACCUCACUCUUCCGUU 1989 D-14821432-1450 1315-1333 UGGCGCUUUCUGCAGUUUGUU 487 CAAACUGCAGAAAGCGCCAUU 1990D-1483 1433-1451 1316-1334 GGCGCUUUCUGCAGUUUGCUU 488GCAAACUGCAGAAAGCGCCUU 1991 D-1484 1434-1452 1317-1335GCGCUUUCUGCAGUUUGCAUU 489 UGCAAACUGCAGAAAGCGCUU 1992 D-1485 1435-14531318-1336 CGCUUUCUGCAGUUUGCAGUU 490 CUGCAAACUGCAGAAAGCGUU 1993 D-14861436-1454 1319-1337 GCUUUCUGCAGUUUGCAGGUU 491 CCUGCAAACUGCAGAAAGCUU 1994D-1487 1437-1455 1320-1338 CUUUCUGCAGUUUGCAGGUUU 492ACCUGCAAACUGCAGAAAGUU 1995 D-1488 1438-1456 1321-1339UUUCUGCAGUUUGCAGGUUUU 493 AACCUGCAAACUGCAGAAAUU 1996 D-1489 1439-14571322-1340 UUCUGCAGUUUGCAGGUUAUU 494 UAACCUGCAAACUGCAGAAUU 1997 D-14901440-1458 1323-1341 UCUGCAGUUUGCAGGUUAUUU 495 AUAACCUGCAAACUGCAGAUU 1998D-1491 1441-1459 1324-1342 CUGCAGUUUGCAGGUUAUCUU 496GAUAACCUGCAAACUGCAGUU 1999 D-1492 144-162 144-162 UACUGUCCAAAGUCCCGGGUU497 CCCGGGACUUUGGACAGUAUU 2000 D-1493 1442-1460 1325-1343UGCAGUUUGCAGGUUAUCAUU 498 UGAUAACCUGCAAACUGCAUU 2001 D-1494 1443-14611326-1344 GCAGUUUGCAGGUUAUCAUUU 499 AUGAUAACCUGCAAACUGCUU 2002 D-14951444-1462 1327-1345 CAGUUUGCAGGUUAUCAUUUU 500 AAUGAUAACCUGCAAACUGUU 2003D-1496 1445-1463 1328-1346 AGUUUGCAGGUUAUCAUUGUU 501CAAUGAUAACCUGCAAACUUU 2004 D-1497 1446-1464 1329-1347GUUUGCAGGUUAUCAUUGUUU 502 ACAAUGAUAACCUGCAAACUU 2005 D-1498 1447-14651330-1348 UUUGCAGGUUAUCAUUGUGUU 503 CACAAUGAUAACCUGCAAAUU 2006 D-14991448-1466 1331-1349 UUGCAGGUUAUCAUUGUGAUU 504 UCACAAUGAUAACCUGCAAUU 2007D-1500 1449-1467 1332-1350 UGCAGGUUAUCAUUGUGAAUU 505UUCACAAUGAUAACCUGCAUU 2008 D-1501 1450-1468 1333-1351GCAGGUUAUCAUUGUGAACUU 506 GUUCACAAUGAUAACCUGCUU 2009 D-1502 1451-14691334-1352 CAGGUUAUCAUUGUGAACUUU 507 AGUUCACAAUGAUAACCUGUU 2010 D-1503145-163 145-163 ACUGUCCAAAGUCCCGGGCUU 508 GCCCGGGACUUUGGACAGUUU 2011D-1504 1452-1470 1335-1353 AGGUUAUCAUUGUGAACUUUU 509AAGUUCACAAUGAUAACCUUU 2012 D-1505 1453-1471 1336-1354GGUUAUCAUUGUGAACUUUUU 510 AAAGUUCACAAUGAUAACCUU 2013 D-1506 1454-14721337-1355 GUUAUCAUUGUGAACUUUUUU 511 AAAAGUUCACAAUGAUAACUU 2014 D-15071455-1473 1338-1356 UUAUCAUUGUGAACUUUUUUU 512 AAAAAGUUCACAAUGAUAAUU 2015D-1508 1456-1474 1339-1357 UAUCAUUGUGAACUUUUUUUU 513AAAAAAGUUCACAAUGAUAUU 2016 D-1509 1457-1475 1340-1358AUCAUUGUGAACUUUUUUUUU 514 AAAAAAAGUUCACAAUGAUUU 2017 D-1510 1458-14761341-1359 UCAUUGUGAACUUUUUUUUUU 515 AAAAAAAAGUUCACAAUGAUU 2018 D-15111459-1477 1342-1360 CAUUGUGAACUUUUUUUUUUU 516 AAAAAAAAAGUUCACAAUGUU 2019D-1512 1460-1478 1343-1361 AUUGUGAACUUUUUUUUUUUU 517AAAAAAAAAAGUUCACAAUUU 2020 D-1513 1461-1479 1344-1362UUGUGAACUUUUUUUUUUUUU 518 AAAAAAAAAAAGUUCACAAUU 2021 D-1514 146-164146-164 CUGUCCAAAGUCCCGGGCAUU 519 UGCCCGGGACUUUGGACAGUU 2022 D-15151462-1480 1345-1363 UGUGAACUUUUUUUUUUUAUU 520 UAAAAAAAAAAAGUUCACAUU 2023D-1516 1463-1481 1346-1364 GUGAACUUUUUUUUUUUAAUU 521UUAAAAAAAAAAAGUUCACUU 2024 D-1517 1464-1482 1347-1365UGAACUUUUUUUUUUUAAGUU 522 CUUAAAAAAAAAAAGUUCAUU 2025 D-1518 1465-14831348-1366 GAACUUUUUUUUUUUAAGAUU 523 UCUUAAAAAAAAAAAGUUCUU 2026 D-15191466-1484 1349-1367 AACUUUUUUUUUUUAAGAGUU 524 CUCUUAAAAAAAAAAAGUUUU 2027D-1520 1467-1485 1350-1368 ACUUUUUUUUUUUAAGAGUUU 525ACUCUUAAAAAAAAAAAGUUU 2028 D-1521 1468-1486 1351-1369CUUUUUUUUUUUAAGAGUAUU 526 UACUCUUAAAAAAAAAAAGUU 2029 D-1522 1469-14871352-1370 UUUUUUUUUUUAAGAGUAAUU 527 UUACUCUUAAAAAAAAAAAUU 2030 D-15231470-1488 1353-1371 UUUUUUUUUUAAGAGUAAAUU 528 UUUACUCUUAAAAAAAAAAUU 2031D-1524 1471-1489 1354-1372 UUUUUUUUUAAGAGUAAAAUU 529UUUUACUCUUAAAAAAAAAUU 2032 D-1525 147-165 147-165 UGUCCAAAGUCCCGGGCACUU530 GUGCCCGGGACUUUGGACAUU 2033 D-1526 1472-1490 1355-1373UUUUUUUUAAGAGUAAAAAUU 531 UUUUUACUCUUAAAAAAAAUU 2034 D-1527 1473-14911356-1374 UUUUUUUAAGAGUAAAAAGUU 532 CUUUUUACUCUUAAAAAAAUU 2035 D-15281474-1492 1357-1375 UUUUUUAAGAGUAAAAAGAUU 533 UCUUUUUACUCUUAAAAAAUU 2036D-1529 1475-1493 1358-1376 UUUUUAAGAGUAAAAAGAAUU 534UUCUUUUUACUCUUAAAAAUU 2037 D-1530 1476-1494 1359-1377UUUUAAGAGUAAAAAGAAAUU 535 UUUCUUUUUACUCUUAAAAUU 2038 D-1531 1477-14951360-1378 UUUAAGAGUAAAAAGAAAUUU 536 AUUUCUUUUUACUCUUAAAUU 2039 D-15321478-1496 1361-1379 UUAAGAGUAAAAAGAAAUAUU 537 UAUUUCUUUUUACUCUUAAUU 2040D-1533 1479-1497 1362-1380 UAAGAGUAAAAAGAAAUAUUU 538AUAUUUCUUUUUACUCUUAUU 2041 D-1534 1480-1498 1363-1381AAGAGUAAAAAGAAAUAUAUU 539 UAUAUUUCUUUUUACUCUUUU 2042 D-1535 1481-14991364-1382 AGAGUAAAAAGAAAUAUACUU 540 GUAUAUUUCUUUUUACUCUUU 2043 D-1536148-166 148-166 GUCCAAAGUCCCGGGCACUUU 541 AGUGCCCGGGACUUUGGACUU 2044D-1537 1482-1500 1365-1383 GAGUAAAAAGAAAUAUACCUU 542GGUAUAUUUCUUUUUACUCUU 2045 D-1538 1483-1501 — AGUAAAAAGAAAUAUACCUUU 543AGGUAUAUUUCUUUUUACUUU 2046 D-1539 1484-1502 — GUAAAAAGAAAUAUACCUAUU 544UAGGUAUAUUUCUUUUUACUU 2047 D-1540 1485-1503 — UAAAAAGAAAUAUACCUAAUU 545UUAGGUAUAUUUCUUUUUAUU 2048 D-1541 149-167 149-167 UCCAAAGUCCCGGGCACUGUU546 CAGUGCCCGGGACUUUGGAUU 2049 D-1542 150-168 150-168CCAAAGUCCCGGGCACUGGUU 547 CCAGUGCCCGGGACUUUGGUU 2050 D-1543 151-169151-169 CAAAGUCCCGGGCACUGGAUU 548 UCCAGUGCCCGGGACUUUGUU 2051 D-1544152-170 152-170 AAAGUCCCGGGCACUGGAGUU 549 CUCCAGUGCCCGGGACUUUUU 2052D-1545 153-171 153-171 AAGUCCCGGGCACUGGAGAUU 550 UCUCCAGUGCCCGGGACUUUU2053 D-1546 15-33 15-33 GGAAGAGUGAGGUGACUGGUU 551 CCAGUCACCUCACUCUUCCUU2054 D-1547 154-172 154-172 AGUCCCGGGCACUGGAGAUUU 552AUCUCCAGUGCCCGGGACUUU 2055 D-1548 155-173 155-173 GUCCCGGGCACUGGAGAUGUU553 CAUCUCCAGUGCCCGGGACUU 2056 D-1549 156-174 156-174UCCCGGGCACUGGAGAUGCUU 554 GCAUCUCCAGUGCCCGGGAUU 2057 D-1550 157-175157-175 CCCGGGCACUGGAGAUGCCUU 555 GGCAUCUCCAGUGCCCGGGUU 2058 D-1551158-176 158-176 CCGGGCACUGGAGAUGCCAUU 556 UGGCAUCUCCAGUGCCCGGUU 2059D-1552 159-177 159-177 CGGGCACUGGAGAUGCCACUU 557 GUGGCAUCUCCAGUGCCCGUU2060 D-1553 160-178 160-178 GGGCACUGGAGAUGCCACGUU 558CGUGGCAUCUCCAGUGCCCUU 2061 D-1554 161-179 161-179 GGCACUGGAGAUGCCACGUUU559 ACGUGGCAUCUCCAGUGCCUU 2062 D-1555 162-180 162-180GCACUGGAGAUGCCACGUUUU 560 AACGUGGCAUCUCCAGUGCUU 2063 D-1556 163-181163-181 CACUGGAGAUGCCACGUUUUU 561 AAACGUGGCAUCUCCAGUGUU 2064 D-155716-34 16-34 GAAGAGUGAGGUGACUGGCUU 562 GCCAGUCACCUCACUCUUCUU 2065 D-1558164-182 164-182 ACUGGAGAUGCCACGUUUGUU 563 CAAACGUGGCAUCUCCAGUUU 2066D-1559 165-183 165-183 CUGGAGAUGCCACGUUUGGUU 564 CCAAACGUGGCAUCUCCAGUU2067 D-1560 166-184 166-184 UGGAGAUGCCACGUUUGGCUU 565GCCAAACGUGGCAUCUCCAUU 2068 D-1561 167-185 167-185 GGAGAUGCCACGUUUGGCGUU566 CGCCAAACGUGGCAUCUCCUU 2069 D-1562 168-186 168-186GAGAUGCCACGUUUGGCGUUU 567 ACGCCAAACGUGGCAUCUCUU 2070 D-1563 169-187169-187 AGAUGCCACGUUUGGCGUGUU 568 CACGCCAAACGUGGCAUCUUU 2071 D-1564170-188 170-188 GAUGCCACGUUUGGCGUGCUU 569 GCACGCCAAACGUGGCAUCUU 2072D-1565 171-189 171-189 AUGCCACGUUUGGCGUGCUUU 570 AGCACGCCAAACGUGGCAUUU2073 D-1566 172-190 172-190 UGCCACGUUUGGCGUGCUUUU 571AAGCACGCCAAACGUGGCAUU 2074 D-1567 173-191 173-191 GCCACGUUUGGCGUGCUUGUU572 CAAGCACGCCAAACGUGGCUU 2075 D-1568 17-35 17-35 AAGAGUGAGGUGACUGGCAUU573 UGCCAGUCACCUCACUCUUUU 2076 D-1569 174-192 174-192CCACGUUUGGCGUGCUUGGUU 574 CCAAGCACGCCAAACGUGGUU 2077 D-1570 175-193175-193 CACGUUUGGCGUGCUUGGAUU 575 UCCAAGCACGCCAAACGUGUU 2078 D-1571176-194 176-194 ACGUUUGGCGUGCUUGGACUU 576 GUCCAAGCACGCCAAACGUUU 2079D-1572 177-195 177-195 CGUUUGGCGUGCUUGGACAUU 577 UGUCCAAGCACGCCAAACGUU2080 D-1573 178-196 178-196 GUUUGGCGUGCUUGGACACUU 578GUGUCCAAGCACGCCAAACUU 2081 D-1574 179-497 179-197 UUUGGCGUGCUUGGACACAUU579 UGUGUCCAAGCACGCCAAAUU 2082 D-1575 180-198 180-198UUGGCGUGCUUGGACACACUU 580 GUGUGUCCAAGCACGCCAAUU 2083 D-1576 181-199181-199 UGGCGUGCUUGGACACACAUU 581 UGUGUGUCCAAGCACGCCAUU 2084 D-1577182-200 182-200 GGCGUGCUUGGACACACAGUU 582 CUGUGUGUCCAAGCACGCCUU 2085D-1578 183-201 183-201 GCGUGCUUGGACACACAGAUU 583 UCUGUGUGUCCAAGCACGCUU2086 D-1579 18-36 18-36 AGAGUGAGGUGACUGGCAUUU 584 AUGCCAGUCACCUCACUCUUU2087 D-1580 184-202 184-202 CGUGCUUGGACACACAGACUU 585GUCUGUGUGUCCAAGCACGUU 2088 D-1581 185-203 185-203 GUGCUUGGACACACAGACAUU586 UGUCUGUGUGUCCAAGCACUU 2089 D-1582 186-204 186-204UGCUUGGACACACAGACACUU 587 GUGUCUGUGUGUCCAAGCAUU 2090 D-1583 187-205187-205 GCUUGGACACACAGACACGUU 588 CGUGUCUGUGUGUCCAAGCUU 2091 D-1584188-206 188-206 CUUGGACACACAGACACGCUU 589 GCGUGUCUGUGUGUCCAAGUU 2092D-1585 189-207 189-207 UUGGACACACAGACACGCAUU 590 UGCGUGUCUGUGUGUCCAAUU2093 D-1586 190-208 190-208 UGGACACACAGACACGCAGUU 591CUGCGUGUCUGUGUGUCCAUU 2094 D-1587 191-209 191-209 GGACACACAGACACGCAGAUU592 UCUGCGUGUCUGUGUGUCCUU 2095 D-1588 192-210 192-210GACACACAGACACGCAGACUU 593 GUCUGCGUGUCUGUGUGUCUU 2096 D-1589 193-211193-211 ACACACAGACACGCAGACAUU 594 UGUCUGCGUGUCUGUGUGUUU 2097 D-159019-37 19-37 GAGUGAGGUGACUGGCAUGUU 595 CAUGCCAGUCACCUCACUCUU 2098 D-1591194-212 194-212 CACACAGACACGCAGACACUU 596 GUGUCUGCGUGUCUGUGUGUU 2099D-1592 195-213 195-213 ACACAGACACGCAGACACAUU 597 UGUGUCUGCGUGUCUGUGUUU2100 D-1593 196-214 196-214 CACAGACACGCAGACACAGUU 598CUGUGUCUGCGUGUCUGUGUU 2101 D-1594 197-215 197-215 ACAGACACGCAGACACAGAUU599 UCUGUGUCUGCGUGUCUGUUU 2102 D-1595 198-216 198-216CAGACACGCAGACACAGAGUU 600 CUCUGUGUCUGCGUGUCUGUU 2103 D-1596 199-217199-217 AGACACGCAGACACAGAGAUU 601 UCUCUGUGUCUGCGUGUCUUU 2104 D-1597200-218 200-218 GACACGCAGACACAGAGACUU 602 GUCUCUGUGUCUGCGUGUCUU 2105D-1598 201-219 201-219 ACACGCAGACACAGAGACAUU 603 UGUCUCUGUGUCUGCGUGUUU2106 D-1599 202-220 202-220 CACGCAGACACAGAGACACUU 604GUGUCUCUGUGUCUGCGUGUU 2107 D-1600 203-221 203-221 ACGCAGACACAGAGACACCUU605 GGUGUCUCUGUGUCUGCGUUU 2108 D-1601 20-38 20-38 AGUGAGGUGACUGGCAUGUUU606 ACAUGCCAGUCACCUCACUUU 2109 D-1602 204-222 204-222CGCAGACACAGAGACACCGUU 607 CGGUGUCUCUGUGUCUGCGUU 2110 D-1603 205-223205-223 GCAGACACAGAGACACCGGUU 608 CCGGUGUCUCUGUGUCUGCUU 2111 D-1604206-224 206-224 CAGACACAGAGACACCGGGUU 609 CCCGGUGUCUCUGUGUCUGUU 2112D-1605 207-225 207-225 AGACACAGAGACACCGGGGUU 610 CCCCGGUGUCUCUGUGUCUUU2113 D-1606 208-226 208-226 GACACAGAGACACCGGGGCUU 611GCCCCGGUGUCUCUGUGUCUU 2114 D-1607 209-227 209-227 ACACAGAGACACCGGGGCCUU612 GGCCCCGGUGUCUCUGUGUUU 2115 D-1608 210-228 210-228CACAGAGACACCGGGGCCCUU 613 GGGCCCCGGUGUCUCUGUGUU 2116 D-1609 211-229211-229 ACAGAGACACCGGGGCCCAUU 614 UGGGCCCCGGUGUCUCUGUUU 2117 D-1610212-230 212-230 CAGAGACACCGGGGCCCAGUU 615 CUGGGCCCCGGUGUCUCUGUU 2118D-1611 213-231 213-231 AGAGACACCGGGGCCCAGGUU 616 CCUGGGCCCCGGUGUCUCUUU2119 D-1612 21-39 21-39 GUGAGGUGACUGGCAUGUGUU 617 CACAUGCCAGUCACCUCACUU2120 D-1613 214-232 214-232 GAGACACCGGGGCCCAGGGUU 618CCCUGGGCCCCGGUGUCUCUU 2121 D-1614 215-233 215-233 AGACACCGGGGCCCAGGGCUU619 GCCCUGGGCCCCGGUGUCUUU 2122 D-1615 216-234 216-234GACACCGGGGCCCAGGGCCUU 620 GGCCCUGGGCCCCGGUGUCUU 2123 D-1616 217-235217-235 ACACCGGGGCCCAGGGCCCUU 621 GGGCCCUGGGCCCCGGUGUUU 2124 D-1617218-236 218-236 CACCGGGGCCCAGGGCCCUUU 622 AGGGCCCUGGGCCCCGGUGUU 2125D-1618 219-237 219-237 ACCGGGGCCCAGGGCCCUCUU 623 GAGGGCCCUGGGCCCCGGUUU2126 D-1619  2-20  2-20 CCAAACGGUGCACGGAAGAUU 624 UCUUCCGUGCACCGUUUGGUU2127 D-1620 220-238 220-238 CCGGGGCCCAGGGCCCUCCUU 625GGAGGGCCCUGGGCCCCGGUU 2128 D-1621 221-239 221-239 CGGGGCCCAGGGCCCUCCUUU626 AGGAGGGCCCUGGGCCCCGUU 2129 D-1622 222-240 222-240GGGGCCCAGGGCCCUCCUAUU 627 UAGGAGGGCCCUGGGCCCCUU 2130 D-1623 223-241223-241 GGGCCCAGGGCCCUCCUAUUU 628 AUAGGAGGGCCCUGGGCCCUU 2131 D-162422-40 22-40 UGAGGUGACUGGCAUGUGUUU 629 ACACAUGCCAGUCACCUCAUU 2132 D-1625224-242 224-242 GGCCCAGGGCCCUCCUAUGUU 630 CAUAGGAGGGCCCUGGGCCUU 2133D-1626 225-243 225-243 GCCCAGGGCCCUCCUAUGGUU 631 CCAUAGGAGGGCCCUGGGCUU2134 D-1627 226-244 226-244 CCCAGGGCCCUCCUAUGGAUU 632UCCAUAGGAGGGCCCUGGGUU 2135 D-1628 227-245 227-245 CCAGGGCCCUCCUAUGGACUU633 GUCCAUAGGAGGGCCCUGGUU 2136 D-1629 228-246 228-246CAGGGCCCUCCUAUGGACCUU 634 GGUCCAUAGGAGGGCCCUGUU 2137 D-1630 229-247229-247 AGGGCCCUCCUAUGGACCCUU 635 GGGUCCAUAGGAGGGCCCUUU 2138 D-1631230-248 230-248 GGGCCCUCCUAUGGACCCUUU 636 AGGGUCCAUAGGAGGGCCCUU 2139D-1632 231-249 231-249 GGCCCUCCUAUGGACCCUGUU 637 CAGGGUCCAUAGGAGGGCCUU2140 D-1633 232-250 232-250 GCCCUCCUAUGGACCCUGCUU 638GCAGGGUCCAUAGGAGGGCUU 2141 D-1634 233-251 233-251 CCCUCCUAUGGACCCUGCCUU639 GGCAGGGUCCAUAGGAGGGUU 2142 D-1635 23-41 23-41 GAGGUGACUGGCAUGUGUGUU640 CACACAUGCCAGUCACCUCUU 2143 D-1636 234-252 234-252CCUCCUAUGGACCCUGCCCUU 641 GGGCAGGGUCCAUAGGAGGUU 2144 D-1637 235-253235-253 CUCCUAUGGACCCUGCCCGUU 642 CGGGCAGGGUCCAUAGGAGUU 2145 D-1638236-254 236-254 UCCUAUGGACCCUGCCCGCUU 643 GCGGGCAGGGUCCAUAGGAUU 2146D-1639 237-255 237-255 CCUAUGGACCCUGCCCGCUUU 644 AGCGGGCAGGGUCCAUAGGUU2147 D-1640 238-256 238-256 CUAUGGACCCUGCCCGCUCUU 645GAGCGGGCAGGGUCCAUAGUU 2148 D-1641 239-257 239-257 UAUGGACCCUGCCCGCUCCUU646 GGAGCGGGCAGGGUCCAUAUU 2149 D-1642 240-258 240-258AUGGACCCUGCCCGCUCCCUU 647 GGGAGCGGGCAGGGUCCAUUU 2150 D-1643 241-259241-259 UGGACCCUGCCCGCUCCCCUU 648 GGGGAGCGGGCAGGGUCCAUU 2151 D-1644242-260 242-260 GGACCCUGCCCGCUCCCCUUU 649 AGGGGAGCGGGCAGGGUCCUU 2152D-1645 243-261 243-261 GACCCUGCCCGCUCCCCUCUU 650 GAGGGGAGCGGGCAGGGUCUU2153 D-1646 24-42 24-42 AGGUGACUGGCAUGUGUGGUU 651 CCACACAUGCCAGUCACCUUU2154 D-1647 244-262 244-262 ACCCUGCCCGCUCCCCUCCUU 652GGAGGGGAGCGGGCAGGGUUU 2155 D-1648 245-263 245-263 CCCUGCCCGCUCCCCUCCCUU653 GGGAGGGGAGCGGGCAGGGUU 2156 D-1649 246-264 246-264CCUGCCCGCUCCCCUCCCAUU 654 UGGGAGGGGAGCGGGCAGGUU 2157 D-1650 247-265247-265 CUGCCCGCUCCCCUCCCAUUU 655 AUGGGAGGGGAGCGGGCAGUU 2158 D-1651248-266 248-266 UGCCCGCUCCCCUCCCAUUUU 656 AAUGGGAGGGGAGCGGGCAUU 2159D-1652 249-267 249-267 GCCCGCUCCCCUCCCAUUGUU 657 CAAUGGGAGGGGAGCGGGCUU2160 D-1653 250-268 250-268 CCCGCUCCCCUCCCAUUGUUU 658ACAAUGGGAGGGGAGCGGGUU 2161 D-1654 251-269 251-269 CCGCUCCCCUCCCAUUGUCUU659 GACAAUGGGAGGGGAGCGGUU 2162 D-1655 252-270 252-270CGCUCCCCUCCCAUUGUCCUU 660 GGACAAUGGGAGGGGAGCGUU 2163 D-1656 253-271253-271 GCUCCCCUCCCAUUGUCCAUU 661 UGGACAAUGGGAGGGGAGCUU 2164 D-1657254-272 254-272 CUCCCCUCCCAUUGUCCACUU 662 GUGGACAAUGGGAGGGGAGUU 2165D-1658 25-43 25-43 GGUGACUGGCAUGUGUGGGUU 663 CCCACACAUGCCAGUCACCUU 2166D-1659 255-273 255-273 UCCCCUCCCAUUGUCCACGUU 664 CGUGGACAAUGGGAGGGGAUU2167 D-1660 256-274 256-274 CCCCUCCCAUUGUCCACGGUU 665CCGUGGACAAUGGGAGGGGUU 2168 D-1661 257-275 257-275 CCCUCCCAUUGUCCACGGCUU666 GCCGUGGACAAUGGGAGGGUU 2169 D-1662 258-276 258-276CCUCCCAUUGUCCACGGCUUU 667 AGCCGUGGACAAUGGGAGGUU 2170 D-1663 259-277259-277 CUCCCAUUGUCCACGGCUGUU 668 CAGCCGUGGACAAUGGGAGUU 2171 D-1664260-278 260-278 UCCCAUUGUCCACGGCUGUUU 669 ACAGCCGUGGACAAUGGGAUU 2172D-1665 261-279 261-279 CCCAUUGUCCACGGCUGUCUU 670 GACAGCCGUGGACAAUGGGUU2173 D-1666 262-280 262-280 CCAUUGUCCACGGCUGUCCUU 671GGACAGCCGUGGACAAUGGUU 2174 D-1667 263-281 263-281 CAUUGUCCACGGCUGUCCGUU672 CGGACAGCCGUGGACAAUGUU 2175 D-1668 264-282 264-282AUUGUCCACGGCUGUCCGCUU 673 GCGGACAGCCGUGGACAAUUU 2176 D-1669 26-44 26-44GUGACUGGCAUGUGUGGGGUU 674 CCCCACACAUGCCAGUCACUU 2177 D-1670 265-283265-283 UUGUCCACGGCUGUCCGCCUU 675 GGCGGACAGCCGUGGACAAUU 2178 D-1671266-284 266-284 UGUCCACGGCUGUCCGCCCUU 676 GGGCGGACAGCCGUGGACAUU 2179D-1672 267-285 267-285 GUCCACGGCUGUCCGCCCAUU 677 UGGGCGGACAGCCGUGGACUU2180 D-1673 268-286 268-286 UCCACGGCUGUCCGCCCACUU 678GUGGGCGGACAGCCGUGGAUU 2181 D-1674 269-287 269-287 CCACGGCUGUCCGCCCACCUU679 GGUGGGCGGACAGCCGUGGUU 2182 D-1675 270-288 270-288CACGGCUGUCCGCCCACCCUU 680 GGGUGGGCGGACAGCCGUGUU 2183 D-1676 271-289271-289 ACGGCUGUCCGCCCACCCCUU 681 GGGGUGGGCGGACAGCCGUUU 2184 D-1677272-290 272-290 CGGCUGUCCGCCCACCCCCUU 682 GGGGGUGGGCGGACAGCCGUU 2185D-1678 273-291 273-291 GGCUGUCCGCCCACCCCCAUU 683 UGGGGGUGGGCGGACAGCCUU2186 D-1679 274-292 274-292 GCUGUCCGCCCACCCCCAUUU 684AUGGGGGUGGGCGGACAGCUU 2187 D-1680 27-45 27-45 UGACUGGCAUGUGUGGGGGUU 685CCCCCACACAUGCCAGUCAUU 2188 D-1681 275-293 275-293 CUGUCCGCCCACCCCCAUUUU686 AAUGGGGGUGGGCGGACAGUU 2189 D-1682 276-294 276-294UGUCCGCCCACCCCCAUUCUU 687 GAAUGGGGGUGGGCGGACAUU 2190 D-1683 277-295277-295 GUCCGCCCACCCCCAUUCUUU 688 AGAAUGGGGGUGGGCGGACUU 2191 D-1684278-296 278-296 UCCGCCCACCCCCAUUCUCUU 689 GAGAAUGGGGGUGGGCGGAUU 2192D-1685 279-297 279-297 CCGCCCACCCCCAUUCUCCUU 690 GGAGAAUGGGGGUGGGCGGUU2193 D-1686 280-298 280-298 CGCCCACCCCCAUUCUCCAUU 691UGGAGAAUGGGGGUGGGCGUU 2194 D-1687 281-299 281-299 GCCCACCCCCAUUCUCCAAUU692 UUGGAGAAUGGGGGUGGGCUU 2195 D-1688 282-300 282-300CCCACCCCCAUUCUCCAAGUU 693 CUUGGAGAAUGGGGGUGGGUU 2196 D-1689 283-301283-301 CCACCCCCAUUCUCCAAGCUU 694 GCUUGGAGAAUGGGGGUGGUU 2197 D-1690284-302 284-302 CACCCCCAUUCUCCAAGCUUU 695 AGCUUGGAGAAUGGGGGUGUU 2198D-1691 28-46 28-46 GACUGGCAUGUGUGGGGGCUU 696 GCCCCCACACAUGCCAGUCUU 2199D-1692 285-303 285-303 ACCCCCAUUCUCCAAGCUUUU 697 AAGCUUGGAGAAUGGGGGUUU2200 D-1693 286-304 286-304 CCCCCAUUCUCCAAGCUUCUU 698GAAGCUUGGAGAAUGGGGGUU 2201 D-1694 287-305 287-305 CCCCAUUCUCCAAGCUUCAUU699 UGAAGCUUGGAGAAUGGGGUU 2202 D-1695 288-306 288-306CCCAUUCUCCAAGCUUCAGUU 700 CUGAAGCUUGGAGAAUGGGUU 2203 D-1696 289-307289-307 CCAUUCUCCAAGCUUCAGCUU 701 GCUGAAGCUUGGAGAAUGGUU 2204 D-1697290-308 290-308 CAUUCUCCAAGCUUCAGCCUU 702 GGCUGAAGCUUGGAGAAUGUU 2205D-1698 291-309 291-309 AUUCUCCAAGCUUCAGCCCUU 703 GGGCUGAAGCUUGGAGAAUUU2206 D-1699 292-310 292-310 UUCUCCAAGCUUCAGCCCCUU 704GGGGCUGAAGCUUGGAGAAUU 2207 D-1700 293-311 293-311 UCUCCAAGCUUCAGCCCCCUU705 GGGGGCUGAAGCUUGGAGAUU 2208 D-1701 294-312 294-312CUCCAAGCUUCAGCCCCCUUU 706 AGGGGGCUGAAGCUUGGAGUU 2209 D-1702 29-47 29-47ACUGGCAUGUGUGGGGGCAUU 707 UGCCCCCACACAUGCCAGUUU 2210 D-1703 295-313295-313 UCCAAGCUUCAGCCCCCUCUU 708 GAGGGGGCUGAAGCUUGGAUU 2211 D-1704296-314 296-314 CCAAGCUUCAGCCCCCUCCUU 709 GGAGGGGGCUGAAGCUUGGUU 2212D-1705 297-315 297-315 CAAGCUUCAGCCCCCUCCUUU 710 AGGAGGGGGCUGAAGCUUGUU2213 D-1706 298-316 298-316 AAGCUUCAGCCCCCUCCUUUU 711AAGGAGGGGGCUGAAGCUUUU 2214 D-1707 299-317 299-317 AGCUUCAGCCCCCUCCUUAUU712 UAAGGAGGGGGCUGAAGCUUU 2215 D-1708 300-318 300-318GCUUCAGCCCCCUCCUUAGUU 713 CUAAGGAGGGGGCUGAAGCUU 2216 D-1709 301-319301-319 CUUCAGCCCCCUCCUUAGUUU 714 ACUAAGGAGGGGGCUGAAGUU 2217 D-1710302-320 302-320 UUCAGCCCCCUCCUUAGUUUU 715 AACUAAGGAGGGGGCUGAAUU 2218D-1711 303-321 303-321 UCAGCCCCCUCCUUAGUUCUU 716 GAACUAAGGAGGGGGCUGAUU2219 D-1712 304-322 304-322 CAGCCCCCUCCUUAGUUCGUU 717CGAACUAAGGAGGGGGCUGUU 2220 D-1713 30-48 30-48 CUGGCAUGUGUGGGGGCAAUU 718UUGCCCCCACACAUGCCAGUU 2221 D-1714 305-323 305-323 AGCCCCCUCCUUAGUUCGGUU719 CCGAACUAAGGAGGGGGCUUU 2222 D-1715 306-324 306-324GCCCCCUCCUUAGUUCGGCUU 720 GCCGAACUAAGGAGGGGGCUU 2223 D-1716 307-325307-325 CCCCCUCCUUAGUUCGGCAUU 721 UGCCGAACUAAGGAGGGGGUU 2224 D-1717308-326 308-326 CCCCUCCUUAGUUCGGCAUUU 722 AUGCCGAACUAAGGAGGGGUU 2225D-1718 309-327 309-327 CCCUCCUUAGUUCGGCAUCUU 723 GAUGCCGAACUAAGGAGGGUU2226 D-1719 310-328 310-328 CCUCCUUAGUUCGGCAUCUUU 724AGAUGCCGAACUAAGGAGGUU 2227 D-1720 311-329 311-329 CUCCUUAGUUCGGCAUCUGUU725 CAGAUGCCGAACUAAGGAGUU 2228 D-1721 312-330 312-330UCCUUAGUUCGGCAUCUGCUU 726 GCAGAUGCCGAACUAAGGAUU 2229 D-1722 313-331313-331 CCUUAGUUCGGCAUCUGCAUU 727 UGCAGAUGCCGAACUAAGGUU 2230 D-1723314-332 314-332 CUUAGUUCGGCAUCUGCACUU 728 GUGCAGAUGCCGAACUAAGUU 2231D-1724 31-49 31-49 UGGCAUGUGUGGGGGCAACUU 729 GUUGCCCCCACACAUGCCAUU 2232D-1725 315-333 315-333 UUAGUUCGGCAUCUGCACAUU 730 UGUGCAGAUGCCGAACUAAUU2233 D-1726 316-334 316-334 UAGUUCGGCAUCUGCACAGUU 731CUGUGCAGAUGCCGAACUAUU 2234 D-1727 317-335 317-335 AGUUCGGCAUCUGCACAGCUU732 GCUGUGCAGAUGCCGAACUUU 2235 D-1728 318-336 318-336GUUCGGCAUCUGCACAGCAUU 733 UGCUGUGCAGAUGCCGAACUU 2236 D-1729 319-337319-337 UUCGGCAUCUGCACAGCACUU 734 GUGCUGUGCAGAUGCCGAAUU 2237 D-1730320-338 320-338 UCGGCAUCUGCACAGCACUUU 735 AGUGCUGUGCAGAUGCCGAUU 2238D-1731  3-21  3-21 CAAACGGUGCACGGAAGAGUU 736 CUCUUCCGUGCACCGUUUGUU 2239D-1732 321-339 321-339 CGGCAUCUGCACAGCACUGUU 737 CAGUGCUGUGCAGAUGCCGUU2240 D-1733 322-340 322-340 GGCAUCUGCACAGCACUGAUU 738UCAGUGCUGUGCAGAUGCCUU 2241 D-1734 323-341 323-341 GCAUCUGCACAGCACUGAAUU739 UUCAGUGCUGUGCAGAUGCUU 2242 D-1735 324-342 324-342CAUCUGCACAGCACUGAAGUU 740 CUUCAGUGCUGUGCAGAUGUU 2243 D-1736 32-50 32-50GGCAUGUGUGGGGGCAACAUU 741 UGUUGCCCCCACACAUGCCUU 2244 D-1737 325-343325-343 AUCUGCACAGCACUGAAGAUU 742 UCUUCAGUGCUGUGCAGAUUU 2245 D-1738326-344 326-344 UCUGCACAGCACUGAAGAAUU 743 UUCUUCAGUGCUGUGCAGAUU 2246D-1739 327-345 327-345 CUGCACAGCACUGAAGAACUU 744 GUUCUUCAGUGCUGUGCAGUU2247 D-1740 328-346 328-346 UGCACAGCACUGAAGAACCUU 745GGUUCUUCAGUGCUGUGCAUU 2248 D-1741 329-347 329-347 GCACAGCACUGAAGAACCUUU746 AGGUUCUUCAGUGCUGUGCUU 2249 D-1742 330-348 330-348CACAGCACUGAAGAACCUGUU 747 CAGGUUCUUCAGUGCUGUGUU 2250 D-1743 331-349331-349 ACAGCACUGAAGAACCUGGUU 748 CCAGGUUCUUCAGUGCUGUUU 2251 D-1744332-350 332-350 CAGCACUGAAGAACCUGGGUU 749 CCCAGGUUCUUCAGUGCUGUU 2252D-1745 333-351 333-351 AGCACUGAAGAACCUGGGAUU 750 UCCCAGGUUCUUCAGUGCUUU2253 D-1746 334-352 334-352 GCACUGAAGAACCUGGGAAUU 751UUCCCAGGUUCUUCAGUGCUU 2254 D-1747 33-51 33-51 GCAUGUGUGGGGGCAACACUU 752GUGUUGCCCCCACACAUGCUU 2255 D-1748 335-353 335-353 CACUGAAGAACCUGGGAAUUU753 AUUCCCAGGUUCUUCAGUGUU 2256 D-1749 336-354 336-354ACUGAAGAACCUGGGAAUCUU 754 GAUUCCCAGGUUCUUCAGUUU 2257 D-1750 337-355337-355 CUGAAGAACCUGGGAAUCAUU 755 UGAUUCCCAGGUUCUUCAGUU 2258 D-1751338-356 338-356 UGAAGAACCUGGGAAUCAGUU 756 CUGAUUCCCAGGUUCUUCAUU 2259D-1752 339-357 339-357 GAAGAACCUGGGAAUCAGAUU 757 UCUGAUUCCCAGGUUCUUCUU2260 D-1753 340-358 340-358 AAGAACCUGGGAAUCAGACUU 758GUCUGAUUCCCAGGUUCUUUU 2261 D-1754 341-359 341-359 AGAACCUGGGAAUCAGACCUU759 GGUCUGAUUCCCAGGUUCUUU 2262 D-1755 342-360 342-360GAACCUGGGAAUCAGACCCUU 760 GGGUCUGAUUCCCAGGUUCUU 2263 D-1756 343-361343-361 AACCUGGGAAUCAGACCCUUU 761 AGGGUCUGAUUCCCAGGUUUU 2264 D-1757344-362 344-362 ACCUGGGAAUCAGACCCUGUU 762 CAGGGUCUGAUUCCCAGGUUU 2265D-1758 34-52 34-52 CAUGUGUGGGGGCAACACGUU 763 CGUGUUGCCCCCACACAUGUU 2266D-1759 345-363 345-363 CCUGGGAAUCAGACCCUGAUU 764 UCAGGGUCUGAUUCCCAGGUU2267 D-1760 346-364 346-364 CUGGGAAUCAGACCCUGAGUU 765CUCAGGGUCUGAUUCCCAGUU 2268 D-1761 347-365 347-365 UGGGAAUCAGACCCUGAGAUU766 UCUCAGGGUCUGAUUCCCAUU 2269 D-1762 348-366 348-366GGGAAUCAGACCCUGAGACUU 767 GUCUCAGGGUCUGAUUCCCUU 2270 D-1763 349-367349-367 GGAAUCAGACCCUGAGACCUU 768 GGUCUCAGGGUCUGAUUCCUU 2271 D-1764350-368 350-368 GAAUCAGACCCUGAGACCCUU 769 GGGUCUCAGGGUCUGAUUCUU 2272D-1765 351-369 351-369 AAUCAGACCCUGAGACCCUUU 770 AGGGUCUCAGGGUCUGAUUUU2273 D-1766 352-370 352-370 AUCAGACCCUGAGACCCUGUU 771CAGGGUCUCAGGGUCUGAUUU 2274 D-1767 353-371 353-371 UCAGACCCUGAGACCCUGAUU772 UCAGGGUCUCAGGGUCUGAUU 2275 D-1768 354-372 354-372CAGACCCUGAGACCCUGAGUU 773 CUCAGGGUCUCAGGGUCUGUU 2276 D-1769 35-53 35-53AUGUGUGGGGGCAACACGAUU 774 UCGUGUUGCCCCCACACAUUU 2277 D-1770 355-373355-373 AGACCCUGAGACCCUGAGCUU 775 GCUCAGGGUCUCAGGGUCUUU 2278 D-1771356-374 356-374 GACCCUGAGACCCUGAGCAUU 776 UGCUCAGGGUCUCAGGGUCUU 2279D-1772 357-375 357-375 ACCCUGAGACCCUGAGCAAUU 777 UUGCUCAGGGUCUCAGGGUUU2280 D-1773 358-376 358-376 CCCUGAGACCCUGAGCAAUUU 778AUUGCUCAGGGUCUCAGGGUU 2281 D-1774 359-377 359-377 CCUGAGACCCUGAGCAAUCUU779 GAUUGCUCAGGGUCUCAGGUU 2282 D-1775 360-378 360-378CUGAGACCCUGAGCAAUCCUU 780 GGAUUGCUCAGGGUCUCAGUU 2283 D-1776 361-379361-379 UGAGACCCUGAGCAAUCCCUU 781 GGGAUUGCUCAGGGUCUCAUU 2284 D-1777362-380 362-380 GAGACCCUGAGCAAUCCCAUU 782 UGGGAUUGCUCAGGGUCUCUU 2285D-1778 363-381 363-381 AGACCCUGAGCAAUCCCAGUU 783 CUGGGAUUGCUCAGGGUCUUU2286 D-1779 364-382 364-382 GACCCUGAGCAAUCCCAGGUU 784CCUGGGAUUGCUCAGGGUCUU 2287 D-1780 365-383 365-383 ACCCUGAGCAAUCCCAGGUUU785 ACCUGGGAUUGCUCAGGGUUU 2288 D-1781 36-54 36-54 UGUGUGGGGGCAACACGAUUU786 AUCGUGUUGCCCCCACACAUU 2289 D-1782 366-384 366-384CCCUGAGCAAUCCCAGGUCUU 787 GACCUGGGAUUGCUCAGGGUU 2290 D-1783 367-385367-385 CCUGAGCAAUCCCAGGUCCUU 788 GGACCUGGGAUUGCUCAGGUU 2291 D-1784368-386 368-386 CUGAGCAAUCCCAGGUCCAUU 789 UGGACCUGGGAUUGCUCAGUU 2292D-1785 369-387 369-387 UGAGCAAUCCCAGGUCCAGUU 790 CUGGACCUGGGAUUGCUCAUU2293 D-1786 370-388 370-388 GAGCAAUCCCAGGUCCAGCUU 791GCUGGACCUGGGAUUGCUCUU 2294 D-1787 371-389 371-389 AGCAAUCCCAGGUCCAGCGUU792 CGCUGGACCUGGGAUUGCUUU 2295 D-1788 372-390 372-390GCAAUCCCAGGUCCAGCGCUU 793 GCGCUGGACCUGGGAUUGCUU 2296 D-1789 373-391373-391 CAAUCCCAGGUCCAGCGCCUU 794 GGCGCUGGACCUGGGAUUGUU 2297 D-1790374-392 374-392 AAUCCCAGGUCCAGCGCCAUU 795 UGGCGCUGGACCUGGGAUUUU 2298D-1791 375-393 375-393 AUCCCAGGUCCAGCGCCAGUU 796 CUGGCGCUGGACCUGGGAUUU2299 D-1792 37-55 37-55 GUGUGGGGGCAACACGAUUUU 797 AAUCGUGUUGCCCCCACACUU2300 D-1793 376-394 376-394 UCCCAGGUCCAGCGCCAGCUU 798GCUGGCGCUGGACCUGGGAUU 2301 D-1794 377-395 377-395 CCCAGGUCCAGCGCCAGCCUU799 GGCUGGCGCUGGACCUGGGUU 2302 D-1795 378-396 378-396CCAGGUCCAGCGCCAGCCCUU 800 GGGCUGGCGCUGGACCUGGUU 2303 D-1796 379-397379-397 CAGGUCCAGCGCCAGCCCUUU 801 AGGGCUGGCGCUGGACCUGUU 2304 D-1797380-398 380-398 AGGUCCAGCGCCAGCCCUAUU 802 UAGGGCUGGCGCUGGACCUUU 2305D-1798 381-399 381-399 GGUCCAGCGCCAGCCCUAUUU 803 AUAGGGCUGGCGCUGGACCUU2306 D-1799 382-400 382-400 GUCCAGCGCCAGCCCUAUCUU 804GAUAGGGCUGGCGCUGGACUU 2307 D-1800 383-401 383-401 UCCAGCGCCAGCCCUAUCAUU805 UGAUAGGGCUGGCGCUGGAUU 2308 D-1801 384-402 384-402CCAGCGCCAGCCCUAUCAUUU 806 AUGAUAGGGCUGGCGCUGGUU 2309 D-1802 385-403385-403 CAGCGCCAGCCCUAUCAUGUU 807 CAUGAUAGGGCUGGCGCUGUU 2310 D-180338-56 38-56 UGUGGGGGCAACACGAUUCUU 808 GAAUCGUGUUGCCCCCACAUU 2311 D-1804386-404 386-404 AGCGCCAGCCCUAUCAUGAUU 809 UCAUGAUAGGGCUGGCGCUUU 2312D-1805 387-405 387-405 GCGCCAGCCCUAUCAUGACUU 810 GUCAUGAUAGGGCUGGCGCUU2313 D-1806 388-406 388-406 CGCCAGCCCUAUCAUGACCUU 811GGUCAUGAUAGGGCUGGCGUU 2314 D-1807 389-407 389-407 GCCAGCCCUAUCAUGACCAUU812 UGGUCAUGAUAGGGCUGGCUU 2315 D-1808 390-408 390-408CCAGCCCUAUCAUGACCAAUU 813 UUGGUCAUGAUAGGGCUGGUU 2316 D-1809 391-409391-409 CAGCCCUAUCAUGACCAAGUU 814 CUUGGUCAUGAUAGGGCUGUU 2317 D-1810392-410 392-410 AGCCCUAUCAUGACCAAGGUU 815 CCUUGGUCAUGAUAGGGCUUU 2318D-1811 393-411 393-411 GCCCUAUCAUGACCAAGGAUU 816 UCCUUGGUCAUGAUAGGGCUU2319 D-1812 394-412 394-412 CCCUAUCAUGACCAAGGAGUU 817CUCCUUGGUCAUGAUAGGGUU 2320 D-1813 395-413 395-413 CCUAUCAUGACCAAGGAGUUU818 ACUCCUUGGUCAUGAUAGGUU 2321 D-1814 39-57 39-57 GUGGGGGCAACACGAUUCUUU819 AGAAUCGUGUUGCCCCCACUU 2322 D-1815 396-414 396-414CUAUCAUGACCAAGGAGUAUU 820 UACUCCUUGGUCAUGAUAGUU 2323 D-1816 397-415397-415 UAUCAUGACCAAGGAGUAUUU 821 AUACUCCUUGGUCAUGAUAUU 2324 D-1817398-416 398-416 AUCAUGACCAAGGAGUAUCUU 822 GAUACUCCUUGGUCAUGAUUU 2325D-1818 399-417 399-417 UCAUGACCAAGGAGUAUCAUU 823 UGAUACUCCUUGGUCAUGAUU2326 D-1819 400-418 400-418 CAUGACCAAGGAGUAUCAAUU 824UUGAUACUCCUUGGUCAUGUU 2327 D-1820 401-419 401-419 AUGACCAAGGAGUAUCAAGUU825 CUUGAUACUCCUUGGUCAUUU 2328 D-1821 402-420 402-420UGACCAAGGAGUAUCAAGAUU 826 UCUUGAUACUCCUUGGUCAUU 2329 D-1822 403-421403-421 GACCAAGGAGUAUCAAGACUU 827 GUCUUGAUACUCCUUGGUCUU 2330 D-1823404-422 404-422 ACCAAGGAGUAUCAAGACCUU 828 GGUCUUGAUACUCCUUGGUUU 2331D-1824 405-423 405-423 CCAAGGAGUAUCAAGACCUUU 829 AGGUCUUGAUACUCCUUGGUU2332 D-1825 40-58 40-58 UGGGGGCAACACGAUUCUCUU 830 GAGAAUCGUGUUGCCCCCAUU2333 D-1826 406-424 406-424 CAAGGAGUAUCAAGACCUUUU 831AAGGUCUUGAUACUCCUUGUU 2334 D-1827 407-425 407-425 AAGGAGUAUCAAGACCUUCUU832 GAAGGUCUUGAUACUCCUUUU 2335 D-1828 408-426 408-426AGGAGUAUCAAGACCUUCAUU 833 UGAAGGUCUUGAUACUCCUUU 2336 D-1829 409-427409-427 GGAGUAUCAAGACCUUCAGUU 834 CUGAAGGUCUUGAUACUCCUU 2337 D-1830410-428 410-428 GAGUAUCAAGACCUUCAGCUU 835 GCUGAAGGUCUUGAUACUCUU 2338D-1831 411-429 411-429 AGUAUCAAGACCUUCAGCAUU 836 UGCUGAAGGUCUUGAUACUUU2339 D-1832 412-430 412-430 GUAUCAAGACCUUCAGCAUUU 837AUGCUGAAGGUCUUGAUACUU 2340 D-1833 413-431 413-431 UAUCAAGACCUUCAGCAUCUU838 GAUGCUGAAGGUCUUGAUAUU 2341 D-1834 414-432 414-432AUCAAGACCUUCAGCAUCUUU 839 AGAUGCUGAAGGUCUUGAUUU 2342 D-1835 415-433415-433 UCAAGACCUUCAGCAUCUGUU 840 CAGAUGCUGAAGGUCUUGAUU 2343 D-183641-59 41-59 GGGGGCAACACGAUUCUCCUU 841 GGAGAAUCGUGUUGCCCCCUU 2344 D-1837416-434 416-434 CAAGACCUUCAGCAUCUGGUU 842 CCAGAUGCUGAAGGUCUUGUU 2345D-1838 417-435 417-435 AAGACCUUCAGCAUCUGGAUU 843 UCCAGAUGCUGAAGGUCUUUU2346 D-1839 418-436 418-436 AGACCUUCAGCAUCUGGACUU 844GUCCAGAUGCUGAAGGUCUUU 2347 D-1840 419-437 419-437 GACCUUCAGCAUCUGGACAUU845 UGUCCAGAUGCUGAAGGUCUU 2348 D-1841 420-438 420-438ACCUUCAGCAUCUGGACAAUU 846 UUGUCCAGAUGCUGAAGGUUU 2349 D-1842 421-439421-439 CCUUCAGCAUCUGGACAAUUU 847 AUUGUCCAGAUGCUGAAGGUU 2350 D-1843 4-22  4-22 AAACGGUGCACGGAAGAGUUU 848 ACUCUUCCGUGCACCGUUUUU 2351 D-1844422-440 422-440 CUUCAGCAUCUGGACAAUGUU 849 CAUUGUCCAGAUGCUGAAGUU 2352D-1845 423-441 423-441 UUCAGCAUCUGGACAAUGAUU 850 UCAUUGUCCAGAUGCUGAAUU2353 D-1846 424-442 424-442 UCAGCAUCUGGACAAUGAGUU 851CUCAUUGUCCAGAUGCUGAUU 2354 D-1847 425-443 425-443 CAGCAUCUGGACAAUGAGGUU852 CCUCAUUGUCCAGAUGCUGUU 2355 D-1848 42-60 42-60 GGGGCAACACGAUUCUCCUUU853 AGGAGAAUCGUGUUGCCCCUU 2356 D-1849 426-444 426-444AGCAUCUGGACAAUGAGGAUU 854 UCCUCAUUGUCCAGAUGCUUU 2357 D-1850 427-445427-445 GCAUCUGGACAAUGAGGAGUU 855 CUCCUCAUUGUCCAGAUGCUU 2358 D-1851428-446 428-446 CAUCUGGACAAUGAGGAGAUU 856 UCUCCUCAUUGUCCAGAUGUU 2359D-1852 429-447 429-447 AUCUGGACAAUGAGGAGAGUU 857 CUCUCCUCAUUGUCCAGAUUU2360 D-1853 430-448 430-448 UCUGGACAAUGAGGAGAGUUU 858ACUCUCCUCAUUGUCCAGAUU 2361 D-1854 431-449 431-449 CUGGACAAUGAGGAGAGUGUU859 CACUCUCCUCAUUGUCCAGUU 2362 D-1855 432-450 432-450UGGACAAUGAGGAGAGUGAUU 860 UCACUCUCCUCAUUGUCCAUU 2363 D-1856 433-451433-451 GGACAAUGAGGAGAGUGACUU 861 GUCACUCUCCUCAUUGUCCUU 2364 D-1857434-452 434-452 GACAAUGAGGAGAGUGACCUU 862 GGUCACUCUCCUCAUUGUCUU 2365D-1858 435-453 435-453 ACAAUGAGGAGAGUGACCAUU 863 UGGUCACUCUCCUCAUUGUUU2366 D-1859 43-61 43-61 GGGCAACACGAUUCUCCUCUU 864 GAGGAGAAUCGUGUUGCCCUU2367 D-1860 436-454 436-454 CAAUGAGGAGAGUGACCACUU 865GUGGUCACUCUCCUCAUUGUU 2368 D-1861 437-455 437-455 AAUGAGGAGAGUGACCACCUU866 GGUGGUCACUCUCCUCAUUUU 2369 D-1862 438-456 438-456AUGAGGAGAGUGACCACCAUU 867 UGGUGGUCACUCUCCUCAUUU 2370 D-1863 439-457439-457 UGAGGAGAGUGACCACCAUUU 868 AUGGUGGUCACUCUCCUCAUU 2371 D-1864440-458 440-458 GAGGAGAGUGACCACCAUCUU 869 GAUGGUGGUCACUCUCCUCUU 2372D-1865 441-459 441-459 AGGAGAGUGACCACCAUCAUU 870 UGAUGGUGGUCACUCUCCUUU2373 D-1866 442-460 442-460 GGAGAGUGACCACCAUCAGUU 871CUGAUGGUGGUCACUCUCCUU 2374 D-1867 443-461 443-461 GAGAGUGACCACCAUCAGCUU872 GCUGAUGGUGGUCACUCUCUU 2375 D-1868 444-462 444-462AGAGUGACCACCAUCAGCUUU 873 AGCUGAUGGUGGUCACUCUUU 2376 D-1869 445-463445-463 GAGUGACCACCAUCAGCUCUU 874 GAGCUGAUGGUGGUCACUCUU 2377 D-187044-62 44-62 GGCAACACGAUUCUCCUCCUU 875 GGAGGAGAAUCGUGUUGCCUU 2378 D-1871446-464 446-464 AGUGACCACCAUCAGCUCAUU 876 UGAGCUGAUGGUGGUCACUUU 2379D-1872 447-465 447-465 GUGACCACCAUCAGCUCAGUU 877 CUGAGCUGAUGGUGGUCACUU2380 D-1873 448-466 448-466 UGACCACCAUCAGCUCAGAUU 878UCUGAGCUGAUGGUGGUCAUU 2381 D-1874 449-467 449-467 GACCACCAUCAGCUCAGAAUU879 UUCUGAGCUGAUGGUGGUCUU 2382 D-1875 450-468 450-468ACCACCAUCAGCUCAGAAAUU 880 UUUCUGAGCUGAUGGUGGUUU 2383 D-1876 451-469451-469 CCACCAUCAGCUCAGAAAAUU 881 UUUUCUGAGCUGAUGGUGGUU 2384 D-1877452-470 452-470 CACCAUCAGCUCAGAAAAGUU 882 CUUUUCUGAGCUGAUGGUGUU 2385D-1878 453-471 — ACCAUCAGCUCAGAAAAGGUU 883 CCUUUUCUGAGCUGAUGGUUU 2386D-1879 454-472 — CCAUCAGCUCAGAAAAGGGUU 884 CCCUUUUCUGAGCUGAUGGUU 2387D-1880 455-473 — CAUCAGCUCAGAAAAGGGCUU 885 GCCCUUUUCUGAGCUGAUGUU 2388D-1881 45-63 45-63 GCAACACGAUUCUCCUCCCUU 886 GGGAGGAGAAUCGUGUUGCUU 2389D-1882 456-474 — AUCAGCUCAGAAAAGGGCCUU 887 GGCCCUUUUCUGAGCUGAUUU 2390D-1883 457-475 — UCAGCUCAGAAAAGGGCCAUU 888 UGGCCCUUUUCUGAGCUGAUU 2391D-1884 458-476 — CAGCUCAGAAAAGGGCCACUU 889 GUGGCCCUUUUCUGAGCUGUU 2392D-1885 459-477 — AGCUCAGAAAAGGGCCACCUU 890 GGUGGCCCUUUUCUGAGCUUU 2393D-1886 460-478 — GCUCAGAAAAGGGCCACCUUU 891 AGGUGGCCCUUUUCUGAGCUU 2394D-1887 461-479 — CUCAGAAAAGGGCCACCUCUU 892 GAGGUGGCCCUUUUCUGAGUU 2395D-1888 462-480 — UCAGAAAAGGGCCACCUCCUU 893 GGAGGUGGCCCUUUUCUGAUU 2396D-1889 463-481 — CAGAAAAGGGCCACCUCCUUU 894 AGGAGGUGGCCCUUUUCUGUU 2397D-1890 464-482 — AGAAAAGGGCCACCUCCUCUU 895 GAGGAGGUGGCCCUUUUCUUU 2398D-1891 465-483 — GAAAAGGGCCACCUCCUCCUU 896 GGAGGAGGUGGCCCUUUUCUU 2399D-1892 46-64 46-64 CAACACGAUUCUCCUCCCUUU 897 AGGGAGGAGAAUCGUGUUGUU 2400D-1893 466-484 — AAAAGGGCCACCUCCUCCCUU 898 GGGAGGAGGUGGCCCUUUUUU 2401D-1894 467-485 — AAAGGGCCACCUCCUCCCCUU 899 GGGGAGGAGGUGGCCCUUUUU 2402D-1895 468-486 — AAGGGCCACCUCCUCCCCAUU 900 UGGGGAGGAGGUGGCCCUUUU 2403D-1896 469-487 — AGGGCCACCUCCUCCCCAGUU 901 CUGGGGAGGAGGUGGCCCUUU 2404D-1897 470-488 — GGGCCACCUCCUCCCCAGCUU 902 GCUGGGGAGGAGGUGGCCCUU 2405D-1898 471-489 — GGCCACCUCCUCCCCAGCCUU 903 GGCUGGGGAGGAGGUGGCCUU 2406D-1899 472-490 — GCCACCUCCUCCCCAGCCCUU 904 GGGCUGGGGAGGAGGUGGCUU 2407D-1900 473-491 — CCACCUCCUCCCCAGCCCCUU 905 GGGGCUGGGGAGGAGGUGGUU 2408D-1901 474-492 — CACCUCCUCCCCAGCCCCUUU 906 AGGGGCUGGGGAGGAGGUGUU 2409D-1902 475-493 — ACCUCCUCCCCAGCCCCUCUU 907 GAGGGGCUGGGGAGGAGGUUU 2410D-1903 476-494 — CCUCCUCCCCAGCCCCUCCUU 908 GGAGGGGCUGGGGAGGAGGUU 2411D-1904 47-65 47-65 AACACGAUUCUCCUCCCUGUU 909 CAGGGAGGAGAAUCGUGUUUU 2412D-1905 477-495 — CUCCUCCCCAGCCCCUCCUUU 910 AGGAGGGGCUGGGGAGGAGUU 2413D-1906 478-496 — UCCUCCCCAGCCCCUCCUGUU 911 CAGGAGGGGCUGGGGAGGAUU 2414D-1907 479-497 — CCUCCCCAGCCCCUCCUGCUU 912 GCAGGAGGGGCUGGGGAGGUU 2415D-1908 480-498 — CUCCCCAGCCCCUCCUGCAUU 913 UGCAGGAGGGGCUGGGGAGUU 2416D-1909 481-499 — UCCCCAGCCCCUCCUGCAGUU 914 CUGCAGGAGGGGCUGGGGAUU 2417D-1910 482-500 — CCCCAGCCCCUCCUGCAGCUU 915 GCUGCAGGAGGGGCUGGGGUU 2418D-1911 483-501 — CCCAGCCCCUCCUGCAGCGUU 916 CGCUGCAGGAGGGGCUGGGUU 2419D-1912 484-502 — CCAGCCCCUCCUGCAGCGUUU 917 ACGCUGCAGGAGGGGCUGGUU 2420D-1913 485-503 — CAGCCCCUCCUGCAGCGUCUU 918 GACGCUGCAGGAGGGGCUGUU 2421D-1914 486-504 — AGCCCCUCCUGCAGCGUCUUU 919 AGACGCUGCAGGAGGGGCUUU 2422D-1915 48-66 48-66 ACACGAUUCUCCUCCCUGGUU 920 CCAGGGAGGAGAAUCGUGUUU 2423D-1916 487-505 — GCCCCUCCUGCAGCGUCUCUU 921 GAGACGCUGCAGGAGGGGCUU 2424D-1917 488-506 — CCCCUCCUGCAGCGUCUCUUU 922 AGAGACGCUGCAGGAGGGGUU 2425D-1918 489-507 — CCCUCCUGCAGCGUCUCUGUU 923 CAGAGACGCUGCAGGAGGGUU 2426D-1919 490-508 — CCUCCUGCAGCGUCUCUGCUU 924 GCAGAGACGCUGCAGGAGGUU 2427D-1920 491-509 — CUCCUGCAGCGUCUCUGCUUU 925 AGCAGAGACGCUGCAGGAGUU 2428D-1921 492-510 — UCCUGCAGCGUCUCUGCUCUU 926 GAGCAGAGACGCUGCAGGAUU 2429D-1922 493-511 — CCUGCAGCGUCUCUGCUCCUU 927 GGAGCAGAGACGCUGCAGGUU 2430D-1923 494-512 — CUGCAGCGUCUCUGCUCCGUU 928 CGGAGCAGAGACGCUGCAGUU 2431D-1924 495-513 — UGCAGCGUCUCUGCUCCGGUU 929 CCGGAGCAGAGACGCUGCAUU 2432D-1925 496-514 — GCAGCGUCUCUGCUCCGGAUU 930 UCCGGAGCAGAGACGCUGCUU 2433D-1926 49-67 49-67 CACGAUUCUCCUCCCUGGGUU 931 CCCAGGGAGGAGAAUCGUGUU 2434D-1927 497-515 — CAGCGUCUCUGCUCCGGACUU 932 GUCCGGAGCAGAGACGCUGUU 2435D-1928 498-516 — AGCGUCUCUGCUCCGGACCUU 933 GGUCCGGAGCAGAGACGCUUU 2436D-1929 499-517 — GCGUCUCUGCUCCGGACCUUU 934 AGGUCCGGAGCAGAGACGCUU 2437D-1930 500-518 — CGUCUCUGCUCCGGACCUCUU 935 GAGGUCCGGAGCAGAGACGUU 2438D-1931 501-519 — GUCUCUGCUCCGGACCUCGUU 936 CGAGGUCCGGAGCAGAGACUU 2439D-1932 502-520 — UCUCUGCUCCGGACCUCGCUU 937 GCGAGGUCCGGAGCAGAGAUU 2440D-1933 503-521 — CUCUGCUCCGGACCUCGCCUU 938 GGCGAGGUCCGGAGCAGAGUU 2441D-1934 504-522 — UCUGCUCCGGACCUCGCCUUU 939 AGGCGAGGUCCGGAGCAGAUU 2442D-1935 505-523 — CUGCUCCGGACCUCGCCUCUU 940 GAGGCGAGGUCCGGAGCAGUU 2443D-1936 506-524 — UGCUCCGGACCUCGCCUCCUU 941 GGAGGCGAGGUCCGGAGCAUU 2444D-1937 50-68 50-68 ACGAUUCUCCUCCCUGGGGUU 942 CCCCAGGGAGGAGAAUCGUUU 2445D-1938 507-525 — GCUCCGGACCUCGCCUCCUUU 943 AGGAGGCGAGGUCCGGAGCUU 2446D-1939 508-526 — CUCCGGACCUCGCCUCCUCUU 944 GAGGAGGCGAGGUCCGGAGUU 2447D-1940 509-527 — UCCGGACCUCGCCUCCUCCUU 945 GGAGGAGGCGAGGUCCGGAUU 2448D-1941 510-528 — CCGGACCUCGCCUCCUCCUUU 946 AGGAGGAGGCGAGGUCCGGUU 2449D-1942 511-529 — CGGACCUCGCCUCCUCCUGUU 947 CAGGAGGAGGCGAGGUCCGUU 2450D-1943 512-530 — GGACCUCGCCUCCUCCUGCUU 948 GCAGGAGGAGGCGAGGUCCUU 2451D-1944 513-531 — GACCUCGCCUCCUCCUGCUUU 949 AGCAGGAGGAGGCGAGGUCUU 2452D-1945 514-532 — ACCUCGCCUCCUCCUGCUCUU 950 GAGCAGGAGGAGGCGAGGUUU 2453D-1946 515-533 — CCUCGCCUCCUCCUGCUCUUU 951 AGAGCAGGAGGAGGCGAGGUU 2454D-1947 516-534 — CUCGCCUCCUCCUGCUCUCUU 952 GAGAGCAGGAGGAGGCGAGUU 2455D-1948 51-69 51-69 CGAUUCUCCUCCCUGGGGAUU 953 UCCCCAGGGAGGAGAAUCGUU 2456D-1949 517-535 - UCGCCUCCUCCUGCUCUCCUU 954 GGAGAGCAGGAGGAGGCGAUU 2457D-1950 518-536 — CGCCUCCUCCUGCUCUCCCUU 955 GGGAGAGCAGGAGGAGGCGUU 2458D-1951 519-537 — GCCUCCUCCUGCUCUCCCUUU 956 AGGGAGAGCAGGAGGAGGCUU 2459D-1952 520-538 — CCUCCUCCUGCUCUCCCUGUU 957 CAGGGAGAGCAGGAGGAGGUU 2460D-1953 521-539 — CUCCUCCUGCUCUCCCUGGUU 958 CCAGGGAGAGCAGGAGGAGUU 2461D-1954 522-540 — UCCUCCUGCUCUCCCUGGGUU 959 CCCAGGGAGAGCAGGAGGAUU 2462D-1955  5-23  5-23 AACGGUGCACGGAAGAGUGUU 960 CACUCUUCCGUGCACCGUUUU 2463D-1956 523-541 — CCUCCUGCUCUCCCUGGGCUU 961 GCCCAGGGAGAGCAGGAGGUU 2464D-1957 524-542 — CUCCUGCUCUCCCUGGGCCUU 962 GGCCCAGGGAGAGCAGGAGUU 2465D-1958 525-543 — UCCUGCUCUCCCUGGGCCUUU 963 AGGCCCAGGGAGAGCAGGAUU 2466D-1959 526-544 — CCUGCUCUCCCUGGGCCUCUU 964 GAGGCCCAGGGAGAGCAGGUU 2467D-1960 52-70 52-70 GAUUCUCCUCCCUGGGGAGUU 965 CUCCCCAGGGAGGAGAAUCUU 2468D-1961 527-545 — CUGCUCUCCCUGGGCCUCAUU 966 UGAGGCCCAGGGAGAGCAGUU 2469D-1962 528-546 — UGCUCUCCCUGGGCCUCAGUU 967 CUGAGGCCCAGGGAGAGCAUU 2470D-1963 529-547 — GCUCUCCCUGGGCCUCAGCUU 968 GCUGAGGCCCAGGGAGAGCUU 2471D-1964 530-548 — CUCUCCCUGGGCCUCAGCCUU 969 GGCUGAGGCCCAGGGAGAGUU 2472D-1965 531-549 — UCUCCCUGGGCCUCAGCCUUU 970 AGGCUGAGGCCCAGGGAGAUU 2473D-1966 532-550 — CUCCCUGGGCCUCAGCCUCUU 971 GAGGCUGAGGCCCAGGGAGUU 2474D-1967 533-551 — UCCCUGGGCCUCAGCCUCCUU 972 GGAGGCUGAGGCCCAGGGAUU 2475D-1968 534-552 — CCCUGGGCCUCAGCCUCCUUU 973 AGGAGGCUGAGGCCCAGGGUU 2476D-1969 535-553 — CCUGGGCCUCAGCCUCCUGUU 974 CAGGAGGCUGAGGCCCAGGUU 2477D-1970 536-554 — CUGGGCCUCAGCCUCCUGCUU 975 GCAGGAGGCUGAGGCCCAGUU 2478D-1971 53-71 53-71 AUUCUCCUCCCUGGGGAGCUU 976 GCUCCCCAGGGAGGAGAAUUU 2479D-1972 537-555 — UGGGCCUCAGCCUCCUGCUUU 977 AGCAGGAGGCUGAGGCCCAUU 2480D-1973 538-556 — GGGCCUCAGCCUCCUGCUGUU 978 CAGCAGGAGGCUGAGGCCCUU 2481D-1974 539-557 — GGCCUCAGCCUCCUGCUGCUU 979 GCAGCAGGAGGCUGAGGCCUU 2482D-1975 540-558 — GCCUCAGCCUCCUGCUGCUUU 980 AGCAGCAGGAGGCUGAGGCUU 2483D-1976 541-559 — CCUCAGCCUCCUGCUGCUUUU 981 AAGCAGCAGGAGGCUGAGGUU 2484D-1977 542-560 — CUCAGCCUCCUGCUGCUUGUU 982 CAAGCAGCAGGAGGCUGAGUU 2485D-1978 543-561 — UCAGCCUCCUGCUGCUUGUUU 983 ACAAGCAGCAGGAGGCUGAUU 2486D-1979 544-562 — CAGCCUCCUGCUGCUUGUGUU 984 CACAAGCAGCAGGAGGCUGUU 2487D-1980 545-563 — AGCCUCCUGCUGCUUGUGGUU 985 CCACAAGCAGCAGGAGGCUUU 2488D-1981 546-564 — GCCUCCUGCUGCUUGUGGUUU 986 ACCACAAGCAGCAGGAGGCUU 2489D-1982 54-72 54-72 UUCUCCUCCCUGGGGAGCAUU 987 UGCUCCCCAGGGAGGAGAAUU 2490D-1983 547-565 — CCUCCUGCUGCUUGUGGUUUU 988 AACCACAAGCAGCAGGAGGUU 2491D-1984 548-566 — CUCCUGCUGCUUGUGGUUGUU 989 CAACCACAAGCAGCAGGAGUU 2492D-1985 549-567 - UCCUGCUGCUUGUGGUUGUUU 990 ACAACCACAAGCAGCAGGAUU 2493D-1986 550-568 — CCUGCUGCUUGUGGUUGUCUU 991 GACAACCACAAGCAGCAGGUU 2494D-1987 551-569 — CUGCUGCUUGUGGUUGUCUUU 992 AGACAACCACAAGCAGCAGUU 2495D-1988 552-570 — UGCUGCUUGUGGUUGUCUGUU 993 CAGACAACCACAAGCAGCAUU 2496D-1989 553-571 — GCUGCUUGUGGUUGUCUGUUU 994 ACAGACAACCACAAGCAGCUU 2497D-1990 554-572 — CUGCUUGUGGUUGUCUGUGUU 995 CACAGACAACCACAAGCAGUU 2498D-1991 555-573 — UGCUUGUGGUUGUCUGUGUUU 996 ACACAGACAACCACAAGCAUU 2499D-1992 556-574 — GCUUGUGGUUGUCUGUGUGUU 997 CACACAGACAACCACAAGCUU 2500D-1993 55-73 55-73 UCUCCUCCCUGGGGAGCAGUU 998 CUGCUCCCCAGGGAGGAGAUU 2501D-1994 557-575 — CUUGUGGUUGUCUGUGUGAUU 999 UCACACAGACAACCACAAGUU 2502D-1995 558-576 — UUGUGGUUGUCUGUGUGAUUU 1000 AUCACACAGACAACCACAAUU 2503D-1996 559-577 — UGUGGUUGUCUGUGUGAUCUU 1001 GAUCACACAGACAACCACAUU 2504D-1997 560-578 — GUGGUUGUCUGUGUGAUCGUU 1002 CGAUCACACAGACAACCACUU 2505D-1998 561-579 — UGGUUGUCUGUGUGAUCGGUU 1003 CCGAUCACACAGACAACCAUU 2506D-1999 562-580 — GGUUGUCUGUGUGAUCGGAUU 1004 UCCGAUCACACAGACAACCUU 2507D-2000 563-581 — GUUGUCUGUGUGAUCGGAUUU 1005 AUCCGAUCACACAGACAACUU 2508D-2001 564-582 — UUGUCUGUGUGAUCGGAUCUU 1006 GAUCCGAUCACACAGACAAUU 2509D-2002 565-583 — UGUCUGUGUGAUCGGAUCCUU 1007 GGAUCCGAUCACACAGACAUU 2510D-2003 566-584 — GUCUGUGUGAUCGGAUCCCUU 1008 GGGAUCCGAUCACACAGACUU 2511D-2004 56-74 56-74 CUCCUCCCUGGGGAGCAGAUU 1009 UCUGCUCCCCAGGGAGGAGUU 2512D-2005 567-585 — UCUGUGUGAUCGGAUCCCAUU 1010 UGGGAUCCGAUCACACAGAUU 2513D-2006 568-586 — CUGUGUGAUCGGAUCCCAAUU 1011 UUGGGAUCCGAUCACACAGUU 2514D-2007 569-587 — UGUGUGAUCGGAUCCCAAAUU 1012 UUUGGGAUCCGAUCACACAUU 2515D-2008 570-588 — GUGUGAUCGGAUCCCAAAAUU 1013 UUUUGGGAUCCGAUCACACUU 2516D-2009 571-589 — UGUGAUCGGAUCCCAAAACUU 1014 GUUUUGGGAUCCGAUCACAUU 2517D-2010 572-590 — GUGAUCGGAUCCCAAAACUUU 1015 AGUUUUGGGAUCCGAUCACUU 2518D-2011 573-591 — UGAUCGGAUCCCAAAACUCUU 1016 GAGUUUUGGGAUCCGAUCAUU 2519D-2012 574-592 — GAUCGGAUCCCAAAACUCCUU 1017 GGAGUUUUGGGAUCCGAUCUU 2520D-2013 575-593 — AUCGGAUCCCAAAACUCCCUU 1018 GGGAGUUUUGGGAUCCGAUUU 2521D-2014 576-594 — UCGGAUCCCAAAACUCCCAUU 1019 UGGGAGUUUUGGGAUCCGAUU 2522D-2015 57-75 57-75 UCCUCCCUGGGGAGCAGAGUU 1020 CUCUGCUCCCCAGGGAGGAUU 2523D-2016 577-595 — CGGAUCCCAAAACUCCCAGUU 1021 CUGGGAGUUUUGGGAUCCGUU 2524D-2017 578-596 — GGAUCCCAAAACUCCCAGCUU 1022 GCUGGGAGUUUUGGGAUCCUU 2525D-2018 579-597 — GAUCCCAAAACUCCCAGCUUU 1023 AGCUGGGAGUUUUGGGAUCUU 2526D-2019 580-598 — AUCCCAAAACUCCCAGCUGUU 1024 CAGCUGGGAGUUUUGGGAUUU 2527D-2020 581-599 — UCCCAAAACUCCCAGCUGCUU 1025 GCAGCUGGGAGUUUUGGGAUU 2528D-2021 582-600 — CCCAAAACUCCCAGCUGCAUU 1026 UGCAGCUGGGAGUUUUGGGUU 2529D-2022 583-601 — CCAAAACUCCCAGCUGCAGUU 1027 CUGCAGCUGGGAGUUUUGGUU 2530D-2023 584-602 — CAAAACUCCCAGCUGCAGGUU 1028 CCUGCAGCUGGGAGUUUUGUU 2531D-2024 585-603 — AAAACUCCCAGCUGCAGGAUU 1029 UCCUGCAGCUGGGAGUUUUUU 2532D-2025 586-604 — AAACUCCCAGCUGCAGGAGUU 1030 CUCCUGCAGCUGGGAGUUUUU 2533D-2026 58-76 58-76 CCUCCCUGGGGAGCAGAGCUU 1031 GCUCUGCUCCCCAGGGAGGUU 2534D-2027 587-605 — AACUCCCAGCUGCAGGAGGUU 1032 CCUCCUGCAGCUGGGAGUUUU 2535D-2028 588-606 471-489 ACUCCCAGCUGCAGGAGGAUU 1033 UCCUCCUGCAGCUGGGAGUUU2536 D-2029 589-607 472-490 CUCCCAGCUGCAGGAGGAGUU 1034CUCCUCCUGCAGCUGGGAGUU 2537 D-2030 590-608 473-491 UCCCAGCUGCAGGAGGAGCUU1035 GCUCCUCCUGCAGCUGGGAUU 2538 D-2031 591-609 474-492CCCAGCUGCAGGAGGAGCUUU 1036 AGCUCCUCCUGCAGCUGGGUU 2539 D-2032 592-610475-493 CCAGCUGCAGGAGGAGCUGUU 1037 CAGCUCCUCCUGCAGCUGGUU 2540 D-2033593-611 476-494 CAGCUGCAGGAGGAGCUGCUU 1038 GCAGCUCCUCCUGCAGCUGUU 2541D-2034 594-612 477-495 AGCUGCAGGAGGAGCUGCGUU 1039 CGCAGCUCCUCCUGCAGCUUU2542 D-2035 595-613 478-496 GCUGCAGGAGGAGCUGCGGUU 1040CCGCAGCUCCUCCUGCAGCUU 2543 D-2036 596-614 479-497 CUGCAGGAGGAGCUGCGGGUU1041 CCCGCAGCUCCUCCUGCAGUU 2544 D-2037 597-615 480-498UGCAGGAGGAGCUGCGGGGUU 1042 CCCCGCAGCUCCUCCUGCAUU 2545 D-2038 59-77 59-77CUCCCUGGGGAGCAGAGCAUU 1043 UGCUCUGCUCCCCAGGGAGUU 2546 D-2039 598-616481-499 GCAGGAGGAGCUGCGGGGCUU 1044 GCCCCGCAGCUCCUCCUGCUU 2547 D-2040599-617 482-500 CAGGAGGAGCUGCGGGGCCUU 1045 GGCCCCGCAGCUCCUCCUGUU 2548D-2041 600-618 483-501 AGGAGGAGCUGCGGGGCCUUU 1046 AGGCCCCGCAGCUCCUCCUUU2549 D-2042 601-619 484-502 GGAGGAGCUGCGGGGCCUGUU 1047CAGGCCCCGCAGCUCCUCCUU 2550 D-2043 602-620 485-503 GAGGAGCUGCGGGGCCUGAUU1048 UCAGGCCCCGCAGCUCCUCUU 2551 D-2044 603-621 486-504AGGAGCUGCGGGGCCUGAGUU 1049 CUCAGGCCCCGCAGCUCCUUU 2552 D-2045 604-622487-505 GGAGCUGCGGGGCCUGAGAUU 1050 UCUCAGGCCCCGCAGCUCCUU 2553 D-2046605-623 488-506 GAGCUGCGGGGCCUGAGAGUU 1051 CUCUCAGGCCCCGCAGCUCUU 2554D-2047 606-624 489-507 AGCUGCGGGGCCUGAGAGAUU 1052 UCUCUCAGGCCCCGCAGCUUU2555 D-2048 607-625 490-508 GCUGCGGGGCCUGAGAGAGUU 1053CUCUCUCAGGCCCCGCAGCUU 2556 D-2049 60-78 60-78 UCCCUGGGGAGCAGAGCAGUU 1054CUGCUCUGCUCCCCAGGGAUU 2557 D-2050 608-626 491-509 CUGCGGGGCCUGAGAGAGAUU1055 UCUCUCUCAGGCCCCGCAGUU 2558 D-2051 609-627 492-510UGCGGGGCCUGAGAGAGACUU 1056 GUCUCUCUCAGGCCCCGCAUU 2559 D-2052 610-628493-511 GCGGGGCCUGAGAGAGACGUU 1057 CGUCUCUCUCAGGCCCCGCUU 2560 D-2053611-629 494-512 CGGGGCCUGAGAGAGACGUUU 1058 ACGUCUCUCUCAGGCCCCGUU 2561D-2054 612-630 495-513 GGGGCCUGAGAGAGACGUUUU 1059 AACGUCUCUCUCAGGCCCCUU2562 D-2055 613-631 496-514 GGGCCUGAGAGAGACGUUCUU 1060GAACGUCUCUCUCAGGCCCUU 2563 D-2056 614-632 497-515 GGCCUGAGAGAGACGUUCAUU1061 UGAACGUCUCUCUCAGGCCUU 2564 D-2057 615-633 498-516GCCUGAGAGAGACGUUCAGUU 1062 CUGAACGUCUCUCUCAGGCUU 2565 D-2058 616-634499-517 CCUGAGAGAGACGUUCAGCUU 1063 GCUGAACGUCUCUCUCAGGUU 2566 D-2059617-635 500-518 CUGAGAGAGACGUUCAGCAUU 1064 UGCUGAACGUCUCUCUCAGUU 2567D-2060 61-79 61-79 CCCUGGGGAGCAGAGCAGAUU 1065 UCUGCUCUGCUCCCCAGGGUU 2568D-2061 618-636 501-519 UGAGAGAGACGUUCAGCAAUU 1066 UUGCUGAACGUCUCUCUCAUU2569 D-2062 619-637 502-520 GAGAGAGACGUUCAGCAACUU 1067GUUGCUGAACGUCUCUCUCUU 2570 D-2063 620-638 503-521 AGAGAGACGUUCAGCAACUUU1068 AGUUGCUGAACGUCUCUCUUU 2571 D-2064 621-639 504-522GAGAGACGUUCAGCAACUUUU 1069 AAGUUGCUGAACGUCUCUCUU 2572 D-2065 622-640505-523 AGAGACGUUCAGCAACUUCUU 1070 GAAGUUGCUGAACGUCUCUUU 2573 D-2066623-641 506-524 GAGACGUUCAGCAACUUCAUU 1071 UGAAGUUGCUGAACGUCUCUU 2574D-2067  6-24  6-24 ACGGUGCACGGAAGAGUGAUU 1072 UCACUCUUCCGUGCACCGUUU 2575D-2068 624-642 507-525 AGACGUUCAGCAACUUCACUU 1073 GUGAAGUUGCUGAACGUCUUU2576 D-2069 625-643 508-526 GACGUUCAGCAACUUCACAUU 1074UGUGAAGUUGCUGAACGUCUU 2577 D-2070 626-644 509-527 ACGUUCAGCAACUUCACAGUU1075 CUGUGAAGUUGCUGAACGUUU 2578 D-2071 627-645 510-528CGUUCAGCAACUUCACAGCUU 1076 GCUGUGAAGUUGCUGAACGUU 2579 D-2072 62-80 62-80CCUGGGGAGCAGAGCAGAGUU 1077 CUCUGCUCUGCUCCCCAGGUU 2580 D-2073 628-646511-529 GUUCAGCAACUUCACAGCGUU 1078 CGCUGUGAAGUUGCUGAACUU 2581 D-2074629-647 512-530 UUCAGCAACUUCACAGCGAUU 1079 UCGCUGUGAAGUUGCUGAAUU 2582D-2075 630-648 513-531 UCAGCAACUUCACAGCGAGUU 1080 CUCGCUGUGAAGUUGCUGAUU2583 D-2076 631-649 514-532 CAGCAACUUCACAGCGAGCUU 1081GCUCGCUGUGAAGUUGCUGUU 2584 D-2077 632-650 515-533 AGCAACUUCACAGCGAGCAUU1082 UGCUCGCUGUGAAGUUGCUUU 2585 D-2078 633-651 516-534GCAACUUCACAGCGAGCACUU 1083 GUGCUCGCUGUGAAGUUGCUU 2586 D-2079 634-652517-535 CAACUUCACAGCGAGCACGUU 1084 CGUGCUCGCUGUGAAGUUGUU 2587 D-2080635-653 518-536 AACUUCACAGCGAGCACGGUU 1085 CCGUGCUCGCUGUGAAGUUUU 2588D-2081 636-654 519-537 ACUUCACAGCGAGCACGGAUU 1086 UCCGUGCUCGCUGUGAAGUUU2589 D-2082 637-655 520-538 CUUCACAGCGAGCACGGAGUU 1087CUCCGUGCUCGCUGUGAAGUU 2590 D-2083 63-81 63-81 CUGGGGAGCAGAGCAGAGGUU 1088CCUCUGCUCUGCUCCCCAGUU 2591 D-2084 638-656 521-539 UUCACAGCGAGCACGGAGGUU1089 CCUCCGUGCUCGCUGUGAAUU 2592 D-2085 639-657 522-540UCACAGCGAGCACGGAGGCUU 1090 GCCUCCGUGCUCGCUGUGAUU 2593 D-2086 640-658523-541 CACAGCGAGCACGGAGGCCUU 1091 GGCCUCCGUGCUCGCUGUGUU 2594 D-2087641-659 524-542 ACAGCGAGCACGGAGGCCCUU 1092 GGGCCUCCGUGCUCGCUGUUU 2595D-2088 642-660 525-543 CAGCGAGCACGGAGGCCCAUU 1093 UGGGCCUCCGUGCUCGCUGUU2596 D-2089 643-661 526-544 AGCGAGCACGGAGGCCCAGUU 1094CUGGGCCUCCGUGCUCGCUUU 2597 D-2090 644-662 527-545 GCGAGCACGGAGGCCCAGGUU1095 CCUGGGCCUCCGUGCUCGCUU 2598 D-2091 645-663 528-546CGAGCACGGAGGCCCAGGUUU 1096 ACCUGGGCCUCCGUGCUCGUU 2599 D-2092 646-664529-547 GAGCACGGAGGCCCAGGUCUU 1097 GACCUGGGCCUCCGUGCUCUU 2600 D-2093647-665 530-548 AGCACGGAGGCCCAGGUCAUU 1098 UGACCUGGGCCUCCGUGCUUU 2601D-2094 64-82 64-82 UGGGGAGCAGAGCAGAGGCUU 1099 GCCUCUGCUCUGCUCCCCAUU 2602D-2095 648-666 531-549 GCACGGAGGCCCAGGUCAAUU 1100 UUGACCUGGGCCUCCGUGCUU2603 D-2096 649-667 532-550 CACGGAGGCCCAGGUCAAGUU 1101CUUGACCUGGGCCUCCGUGUU 2604 D-2097 650-668 533-551 ACGGAGGCCCAGGUCAAGGUU1102 CCUUGACCUGGGCCUCCGUUU 2605 D-2098 651-669 534-552CGGAGGCCCAGGUCAAGGGUU 1103 CCCUUGACCUGGGCCUCCGUU 2606 D-2099 652-670535-553 GGAGGCCCAGGUCAAGGGCUU 1104 GCCCUUGACCUGGGCCUCCUU 2607 D-2100653-671 536-554 GAGGCCCAGGUCAAGGGCUUU 1105 AGCCCUUGACCUGGGCCUCUU 2608D-2101 654-672 537-555 AGGCCCAGGUCAAGGGCUUUU 1106 AAGCCCUUGACCUGGGCCUUU2609 D-2102 655-673 538-556 GGCCCAGGUCAAGGGCUUGUU 1107CAAGCCCUUGACCUGGGCCUU 2610 D-2103 656-674 539-557 GCCCAGGUCAAGGGCUUGAUU1108 UCAAGCCCUUGACCUGGGCUU 2611 D-2104 657-675 540-558CCCAGGUCAAGGGCUUGAGUU 1109 CUCAAGCCCUUGACCUGGGUU 2612 D-2105 65-83 65-83GGGGAGCAGAGCAGAGGCAUU 1110 UGCCUCUGCUCUGCUCCCCUU 2613 D-2106 658-676541-559 CCAGGUCAAGGGCUUGAGCUU 1111 GCUCAAGCCCUUGACCUGGUU 2614 D-2107659-677 542-560 CAGGUCAAGGGCUUGAGCAUU 1112 UGCUCAAGCCCUUGACCUGUU 2615D-2108 660-678 543-561 AGGUCAAGGGCUUGAGCACUU 1113 GUGCUCAAGCCCUUGACCUUU2616 D-2109 661-679 544-562 GGUCAAGGGCUUGAGCACCUU 1114GGUGCUCAAGCCCUUGACCUU 2617 D-2110 662-680 545-563 GUCAAGGGCUUGAGCACCCUU1115 GGGUGCUCAAGCCCUUGACUU 2618 D-2111 663-681 546-564UCAAGGGCUUGAGCACCCAUU 1116 UGGGUGCUCAAGCCCUUGAUU 2619 D-2112 664-682547-565 CAAGGGCUUGAGCACCCAGUU 1117 CUGGGUGCUCAAGCCCUUGUU 2620 D-2113665-683 548-566 AAGGGCUUGAGCACCCAGGUU 1118 CCUGGGUGCUCAAGCCCUUUU 2621D-2114 666-684 549-567 AGGGCUUGAGCACCCAGGGUU 1119 CCCUGGGUGCUCAAGCCCUUU2622 D-2115 667-685 550-568 GGGCUUGAGCACCCAGGGAUU 1120UCCCUGGGUGCUCAAGCCCUU 2623 D-2116 66-84 66-84 GGGAGCAGAGCAGAGGCAAUU 1121UUGCCUCUGCUCUGCUCCCUU 2624 D-2117 668-686 551-569 GGCUUGAGCACCCAGGGAGUU1122 CUCCCUGGGUGCUCAAGCCUU 2625 D-2118 669-687 552-570GCUUGAGCACCCAGGGAGGUU 1123 CCUCCCUGGGUGCUCAAGCUU 2626 D-2119 670-688553-571 CUUGAGCACCCAGGGAGGCUU 1124 GCCUCCCUGGGUGCUCAAGUU 2627 D-2120671-689 554-572 UUGAGCACCCAGGGAGGCAUU 1125 UGCCUCCCUGGGUGCUCAAUU 2628D-2121 672-690 555-573 UGAGCACCCAGGGAGGCAAUU 1126 UUGCCUCCCUGGGUGCUCAUU2629 D-2122 673-691 556-574 GAGCACCCAGGGAGGCAAUUU 1127AUUGCCUCCCUGGGUGCUCUU 2630 D-2123 674-692 557-575 AGCACCCAGGGAGGCAAUGUU1128 CAUUGCCUCCCUGGGUGCUUU 2631 D-2124 675-693 558-576GCACCCAGGGAGGCAAUGUUU 1129 ACAUUGCCUCCCUGGGUGCUU 2632 D-2125 676-694559-577 CACCCAGGGAGGCAAUGUGUU 1130 CACAUUGCCUCCCUGGGUGUU 2633 D-2126677-695 560-578 ACCCAGGGAGGCAAUGUGGUU 1131 CCACAUUGCCUCCCUGGGUUU 2634D-2127 67-85 67-85 GGAGCAGAGCAGAGGCAACUU 1132 GUUGCCUCUGCUCUGCUCCUU 2635D-2128 678-696 561-579 CCCAGGGAGGCAAUGUGGGUU 1133 CCCACAUUGCCUCCCUGGGUU2636 D-2129 679-697 562-580 CCAGGGAGGCAAUGUGGGAUU 1134UCCCACAUUGCCUCCCUGGUU 2637 D-2130 680-698 563-581 CAGGGAGGCAAUGUGGGAAUU1135 UUCCCACAUUGCCUCCCUGUU 2638 D-2131 681-699 564-582AGGGAGGCAAUGUGGGAAGUU 1136 CUUCCCACAUUGCCUCCCUUU 2639 D-2132 682-700565-583 GGGAGGCAAUGUGGGAAGAUU 1137 UCUUCCCACAUUGCCUCCCUU 2640 D-2133683-701 566-584 GGAGGCAAUGUGGGAAGAAUU 1138 UUCUUCCCACAUUGCCUCCUU 2641D-2134 684-702 567-585 GAGGCAAUGUGGGAAGAAAUU 1139 UUUCUUCCCACAUUGCCUCUU2642 D-2135 685-703 568-586 AGGCAAUGUGGGAAGAAAGUU 1140CUUUCUUCCCACAUUGCCUUU 2643 D-2136 686-704 569-587 GGCAAUGUGGGAAGAAAGAUU1141 UCUUUCUUCCCACAUUGCCUU 2644 D-2137 687-705 570-588GCAAUGUGGGAAGAAAGAUUU 1142 AUCUUUCUUCCCACAUUGCUU 2645 D-2138 68-86 68-86GAGCAGAGCAGAGGCAACCUU 1143 GGUUGCCUCUGCUCUGCUCUU 2646 D-2139 688-706571-589 CAAUGUGGGAAGAAAGAUGUU 1144 CAUCUUUCUUCCCACAUUGUU 2647 D-2140689-707 572-590 AAUGUGGGAAGAAAGAUGAUU 1145 UCAUCUUUCUUCCCACAUUUU 2648D-2141 690-708 573-591 AUGUGGGAAGAAAGAUGAAUU 1146 UUCAUCUUUCUUCCCACAUUU2649 D-2142 691-709 574-592 UGUGGGAAGAAAGAUGAAGUU 1147CUUCAUCUUUCUUCCCACAUU 2650 D-2143 692-710 575-593 GUGGGAAGAAAGAUGAAGUUU1148 ACUUCAUCUUUCUUCCCACUU 2651 D-2144 693-711 576-594UGGGAAGAAAGAUGAAGUCUU 1149 GACUUCAUCUUUCUUCCCAUU 2652 D-2145 694-712577-595 GGGAAGAAAGAUGAAGUCGUU 1150 CGACUUCAUCUUUCUUCCCUU 2653 D-2146695-713 578-596 GGAAGAAAGAUGAAGUCGCUU 1151 GCGACUUCAUCUUUCUUCCUU 2654D-2147 696-714 579-597 GAAGAAAGAUGAAGUCGCUUU 1152 AGCGACUUCAUCUUUCUUCUU2655 D-2148 697-715 580-598 AAGAAAGAUGAAGUCGCUAUU 1153UAGCGACUUCAUCUUUCUUUU 2656 D-2149 69-87 69-87 AGCAGAGCAGAGGCAACCCUU 1154GGGUUGCCUCUGCUCUGCUUU 2657 D-2150 698-716 581-599 AGAAAGAUGAAGUCGCUAGUU1155 CUAGCGACUUCAUCUUUCUUU 2658 D-2151 699-717 581-600GAAAGAUGAAGUCGCUAGAUU 1156 UCUAGCGACUUCAUCUUUCUU 2659 D-2152 700-718581-601 AAAGAUGAAGUCGCUAGAGUU 1157 CUCUAGCGACUUCAUCUUUUU 2660 D-2153701-719 581-602 AAGAUGAAGUCGCUAGAGUUU 1158 ACUCUAGCGACUUCAUCUUUU 2661D-2154 702-720 581-603 AGAUGAAGUCGCUAGAGUCUU 1159 GACUCUAGCGACUUCAUCUUU2662 D-2155 703-721 581-604 GAUGAAGUCGCUAGAGUCCUU 1160GGACUCUAGCGACUUCAUCUU 2663 D-2156 704-722 581-605 AUGAAGUCGCUAGAGUCCCUU1161 GGGACUCUAGCGACUUCAUUU 2664 D-2157 705-723 581-606UGAAGUCGCUAGAGUCCCAUU 1162 UGGGACUCUAGCGACUUCAUU 2665 D-2158 706-724581-607 GAAGUCGCUAGAGUCCCAGUU 1163 CUGGGACUCUAGCGACUUCUU 2666 D-2159707-725 581-608 AAGUCGCUAGAGUCCCAGCUU 1164 GCUGGGACUCUAGCGACUUUU 2667D-2160 708-726 581-609 AGUCGCUAGAGUCCCAGCUUU 1165 AGCUGGGACUCUAGCGACUUU2668 D-2161 70-88 70-88 GCAGAGCAGAGGCAACCCAUU 1166 UGGGUUGCCUCUGCUCUGCUU2669 D-2162 709-727 592-610 GUCGCUAGAGUCCCAGCUGUU 1167CAGCUGGGACUCUAGCGACUU 2670 D-2163 710-728 593-611 UCGCUAGAGUCCCAGCUGGUU1168 CCAGCUGGGACUCUAGCGAUU 2671 D-2164 711-729 594-612CGCUAGAGUCCCAGCUGGAUU 1169 UCCAGCUGGGACUCUAGCGUU 2672 D-2165 712-730595-613 GCUAGAGUCCCAGCUGGAGUU 1170 CUCCAGCUGGGACUCUAGCUU 2673 D-2166713-731 596-614 CUAGAGUCCCAGCUGGAGAUU 1171 UCUCCAGCUGGGACUCUAGUU 2674D-2167 714-732 597-615 UAGAGUCCCAGCUGGAGAAUU 1172 UUCUCCAGCUGGGACUCUAUU2675 D-2168 715-733 598-616 AGAGUCCCAGCUGGAGAAAUU 1173UUUCUCCAGCUGGGACUCUUU 2676 D-2169 716-734 599-617 GAGUCCCAGCUGGAGAAACUU1174 GUUUCUCCAGCUGGGACUCUU 2677 D-2170 717-735 600-618AGUCCCAGCUGGAGAAACAUU 1175 UGUUUCUCCAGCUGGGACUUU 2678 D-2171 718-736601-619 GUCCCAGCUGGAGAAACAGUU 1176 CUGUUUCUCCAGCUGGGACUU 2679 D-217271-89 71-89 CAGAGCAGAGGCAACCCAUUU 1177 AUGGGUUGCCUCUGCUCUGUU 2680 D-2173719-737 602-620 UCCCAGCUGGAGAAACAGCUU 1178 GCUGUUUCUCCAGCUGGGAUU 2681D-2174 720-738 603-621 CCCAGCUGGAGAAACAGCAUU 1179 UGCUGUUUCUCCAGCUGGGUU2682 D-2175 721-739 604-622 CCAGCUGGAGAAACAGCAGUU 1180CUGCUGUUUCUCCAGCUGGUU 2683 D-2176 722-740 605-623 CAGCUGGAGAAACAGCAGAUU1181 UCUGCUGUUUCUCCAGCUGUU 2684 D-2177 723-741 606-624AGCUGGAGAAACAGCAGAAUU 1182 UUCUGCUGUUUCUCCAGCUUU 2685 D-2178 724-742607-625 GCUGGAGAAACAGCAGAAGUU 1183 CUUCUGCUGUUUCUCCAGCUU 2686 D-2179 7-25  7-25 CGGUGCACGGAAGAGUGAGUU 1184 CUCACUCUUCCGUGCACCGUU 2687 D-2180725-743 608-626 CUGGAGAAACAGCAGAAGGUU 1185 CCUUCUGCUGUUUCUCCAGUU 2688D-2181 726-744 609-627 UGGAGAAACAGCAGAAGGAUU 1186 UCCUUCUGCUGUUUCUCCAUU2689 D-2182 727-745 610-628 GGAGAAACAGCAGAAGGACUU 1187GUCCUUCUGCUGUUUCUCCUU 2690 D-2183 728-746 611-629 GAGAAACAGCAGAAGGACCUU1188 GGUCCUUCUGCUGUUUCUCUU 2691 D-2184 72-90 72-90 AGAGCAGAGGCAACCCAUCUU1189 GAUGGGUUGCCUCUGCUCUUU 2692 D-2185 729-747 612-630AGAAACAGCAGAAGGACCUUU 1190 AGGUCCUUCUGCUGUUUCUUU 2693 D-2186 730-748613-631 GAAACAGCAGAAGGACCUGUU 1191 CAGGUCCUUCUGCUGUUUCUU 2694 D-2187731-749 614-632 AAACAGCAGAAGGACCUGAUU 1192 UCAGGUCCUUCUGCUGUUUUU 2695D-2188 732-750 615-633 AACAGCAGAAGGACCUGAGUU 1193 CUCAGGUCCUUCUGCUGUUUU2696 D-2189 733-751 616-634 ACAGCAGAAGGACCUGAGUUU 1194ACUCAGGUCCUUCUGCUGUUU 2697 D-2190 734-752 617-635 CAGCAGAAGGACCUGAGUGUU1195 CACUCAGGUCCUUCUGCUGUU 2698 D-2191 735-753 618-636AGCAGAAGGACCUGAGUGAUU 1196 UCACUCAGGUCCUUCUGCUUU 2699 D-2192 736-754619-637 GCAGAAGGACCUGAGUGAAUU 1197 UUCACUCAGGUCCUUCUGCUU 2700 D-2193737-755 620-638 CAGAAGGACCUGAGUGAAGUU 1198 CUUCACUCAGGUCCUUCUGUU 2701D-2194 738-756 621-639 AGAAGGACCUGAGUGAAGAUU 1199 UCUUCACUCAGGUCCUUCUUU2702 D-2195 73-91 73-91 GAGCAGAGGCAACCCAUCCUU 1200 GGAUGGGUUGCCUCUGCUCUU2703 D-2196 739-757 622-640 GAAGGACCUGAGUGAAGAUUU 1201AUCUUCACUCAGGUCCUUCUU 2704 D-2197 740-758 623-641 AAGGACCUGAGUGAAGAUCUU1202 GAUCUUCACUCAGGUCCUUUU 2705 D-2198 741-759 624-642AGGACCUGAGUGAAGAUCAUU 1203 UGAUCUUCACUCAGGUCCUUU 2706 D-2199 742-760625-643 GGACCUGAGUGAAGAUCACUU 1204 GUGAUCUUCACUCAGGUCCUU 2707 D-2200743-761 626-644 GACCUGAGUGAAGAUCACUUU 1205 AGUGAUCUUCACUCAGGUCUU 2708D-2201 744-762 627-645 ACCUGAGUGAAGAUCACUCUU 1206 GAGUGAUCUUCACUCAGGUUU2709 D-2202 745-763 628-646 CCUGAGUGAAGAUCACUCCUU 1207GGAGUGAUCUUCACUCAGGUU 2710 D-2203 746-764 629-647 CUGAGUGAAGAUCACUCCAUU1208 UGGAGUGAUCUUCACUCAGUU 2711 D-2204 747-765 630-648UGAGUGAAGAUCACUCCAGUU 1209 CUGGAGUGAUCUUCACUCAUU 2712 D-2205 748-766631-649 GAGUGAAGAUCACUCCAGCUU 1210 GCUGGAGUGAUCUUCACUCUU 2713 D-220674-92 74-92 AGCAGAGGCAACCCAUCCCUU 1211 GGGAUGGGUUGCCUCUGCUUU 2714 D-2207749-767 632-650 AGUGAAGAUCACUCCAGCCUU 1212 GGCUGGAGUGAUCUUCACUUU 2715D-2208 750-768 633-651 GUGAAGAUCACUCCAGCCUUU 1213 AGGCUGGAGUGAUCUUCACUU2716 D-2209 751-769 634-652 UGAAGAUCACUCCAGCCUGUU 1214CAGGCUGGAGUGAUCUUCAUU 2717 D-2210 752-770 635-653 GAAGAUCACUCCAGCCUGCUU1215 GCAGGCUGGAGUGAUCUUCUU 2718 D-2211 753-771 636-654AAGAUCACUCCAGCCUGCUUU 1216 AGCAGGCUGGAGUGAUCUUUU 2719 D-2212 754-772637-655 AGAUCACUCCAGCCUGCUGUU 1217 CAGCAGGCUGGAGUGAUCUUU 2720 D-2213755-773 638-656 GAUCACUCCAGCCUGCUGCUU 1218 GCAGCAGGCUGGAGUGAUCUU 2721D-2214 756-774 639-657 AUCACUCCAGCCUGCUGCUUU 1219 AGCAGCAGGCUGGAGUGAUUU2722 D-2215 757-775 640-658 UCACUCCAGCCUGCUGCUCUU 1220GAGCAGCAGGCUGGAGUGAUU 2723 D-2216 758-776 641-659 CACUCCAGCCUGCUGCUCCUU1221 GGAGCAGCAGGCUGGAGUGUU 2724 D-2217 75-93 75-93 GCAGAGGCAACCCAUCCCCUU1222 GGGGAUGGGUUGCCUCUGCUU 2725 D-2218 759-777 642-660ACUCCAGCCUGCUGCUCCAUU 1223 UGGAGCAGCAGGCUGGAGUUU 2726 D-2219 760-778643-661 CUCCAGCCUGCUGCUCCACUU 1224 GUGGAGCAGCAGGCUGGAGUU 2727 D-2220761-779 644-662 UCCAGCCUGCUGCUCCACGUU 1225 CGUGGAGCAGCAGGCUGGAUU 2728D-2221 762-780 645-663 CCAGCCUGCUGCUCCACGUUU 1226 ACGUGGAGCAGCAGGCUGGUU2729 D-2222 763-781 646-664 CAGCCUGCUGCUCCACGUGUU 1227CACGUGGAGCAGCAGGCUGUU 2730 D-2223 764-782 647-665 AGCCUGCUGCUCCACGUGAUU1228 UCACGUGGAGCAGCAGGCUUU 2731 D-2224 765-783 648-666GCCUGCUGCUCCACGUGAAUU 1229 UUCACGUGGAGCAGCAGGCUU 2732 D-2225 766-784649-667 CCUGCUGCUCCACGUGAAGUU 1230 CUUCACGUGGAGCAGCAGGUU 2733 D-2226767-785 650-668 CUGCUGCUCCACGUGAAGCUU 1231 GCUUCACGUGGAGCAGCAGUU 2734D-2227 768-786 651-669 UGCUGCUCCACGUGAAGCAUU 1232 UGCUUCACGUGGAGCAGCAUU2735 D-2228 76-94 76-94 CAGAGGCAACCCAUCCCCCUU 1233 GGGGGAUGGGUUGCCUCUGUU2736 D-2229 769-787 652-670 GCUGCUCCACGUGAAGCAGUU 1234CUGCUUCACGUGGAGCAGCUU 2737 D-2230 770-788 653-671 CUGCUCCACGUGAAGCAGUUU1235 ACUGCUUCACGUGGAGCAGUU 2738 D-2231 771-789 654-672UGCUCCACGUGAAGCAGUUUU 1236 AACUGCUUCACGUGGAGCAUU 2739 D-2232 772-790655-673 GCUCCACGUGAAGCAGUUCUU 1237 GAACUGCUUCACGUGGAGCUU 2740 D-2233773-791 656-674 CUCCACGUGAAGCAGUUCGUU 1238 CGAACUGCUUCACGUGGAGUU 2741D-2234 774-792 657-675 UCCACGUGAAGCAGUUCGUUU 1239 ACGAACUGCUUCACGUGGAUU2742 D-2235 775-793 658-676 CCACGUGAAGCAGUUCGUGUU 1240CACGAACUGCUUCACGUGGUU 2743 D-2236 776-794 659-677 CACGUGAAGCAGUUCGUGUUU1241 ACACGAACUGCUUCACGUGUU 2744 D-2237 777-795 660-678ACGUGAAGCAGUUCGUGUCUU 1242 GACACGAACUGCUUCACGUUU 2745 D-2238 778-796661-679 CGUGAAGCAGUUCGUGUCUUU 1243 AGACACGAACUGCUUCACGUU 2746 D-223977-95 77-95 AGAGGCAACCCAUCCCCCAUU 1244 UGGGGGAUGGGUUGCCUCUUU 2747 D-2240779-797 662-680 GUGAAGCAGUUCGUGUCUGUU 1245 CAGACACGAACUGCUUCACUU 2748D-2241 780-798 663-681 UGAAGCAGUUCGUGUCUGAUU 1246 UCAGACACGAACUGCUUCAUU2749 D-2242 781-799 664-682 GAAGCAGUUCGUGUCUGACUU 1247GUCAGACACGAACUGCUUCUU 2750 D-2243 782-800 665-683 AAGCAGUUCGUGUCUGACCUU1248 GGUCAGACACGAACUGCUUUU 2751 D-2244 783-801 666-684AGCAGUUCGUGUCUGACCUUU 1249 AGGUCAGACACGAACUGCUUU 2752 D-2245 784-802667-685 GCAGUUCGUGUCUGACCUGUU 1250 CAGGUCAGACACGAACUGCUU 2753 D-2246785-803 668-686 CAGUUCGUGUCUGACCUGCUU 1251 GCAGGUCAGACACGAACUGUU 2754D-2247 786-804 669-687 AGUUCGUGUCUGACCUGCGUU 1252 CGCAGGUCAGACACGAACUUU2755 D-2248 787-805 670-688 GUUCGUGUCUGACCUGCGGUU 1253CCGCAGGUCAGACACGAACUU 2756 D-2249 788-806 671-689 UUCGUGUCUGACCUGCGGAUU1254 UCCGCAGGUCAGACACGAAUU 2757 D-2250 78-96 78-96 GAGGCAACCCAUCCCCCACUU1255 GUGGGGGAUGGGUUGCCUCUU 2758 D-2251 789-807 672-690UCGUGUCUGACCUGCGGAGUU 1256 CUCCGCAGGUCAGACACGAUU 2759 D-2252 790-808673-691 CGUGUCUGACCUGCGGAGCUU 1257 GCUCCGCAGGUCAGACACGUU 2760 D-2253791-809 674-692 GUGUCUGACCUGCGGAGCCUU 1258 GGCUCCGCAGGUCAGACACUU 2761D-2254 792-810 675-693 UGUCUGACCUGCGGAGCCUUU 1259 AGGCUCCGCAGGUCAGACAUU2762 D-2255 793-811 676-694 GUCUGACCUGCGGAGCCUGUU 1260CAGGCUCCGCAGGUCAGACUU 2763 D-2256 794-812 677-695 UCUGACCUGCGGAGCCUGAUU1261 UCAGGCUCCGCAGGUCAGAUU 2764 D-2257 795-813 678-696CUGACCUGCGGAGCCUGAGUU 1262 CUCAGGCUCCGCAGGUCAGUU 2765 D-2258 796-814679-697 UGACCUGCGGAGCCUGAGCUU 1263 GCUCAGGCUCCGCAGGUCAUU 2766 D-2259797-815 680-698 GACCUGCGGAGCCUGAGCUUU 1264 AGCUCAGGCUCCGCAGGUCUU 2767D-2260 798-816 681-699 ACCUGCGGAGCCUGAGCUGUU 1265 CAGCUCAGGCUCCGCAGGUUU2768 D-2261 79-97 79-97 AGGCAACCCAUCCCCCACUUU 1266 AGUGGGGGAUGGGUUGCCUUU2769 D-2262 799-817 682-700 CCUGCGGAGCCUGAGCUGUUU 1267ACAGCUCAGGCUCCGCAGGUU 2770 D-2263 800-818 683-701 CUGCGGAGCCUGAGCUGUCUU1268 GACAGCUCAGGCUCCGCAGUU 2771 D-2264 801-819 684-702UGCGGAGCCUGAGCUGUCAUU 1269 UGACAGCUCAGGCUCCGCAUU 2772 D-2265 802-820685-703 GCGGAGCCUGAGCUGUCAGUU 1270 CUGACAGCUCAGGCUCCGCUU 2773 D-2266803-821 686-704 CGGAGCCUGAGCUGUCAGAUU 1271 UCUGACAGCUCAGGCUCCGUU 2774D-2267 804-822 687-705 GGAGCCUGAGCUGUCAGAUUU 1272 AUCUGACAGCUCAGGCUCCUU2775 D-2268 805-823 688-706 GAGCCUGAGCUGUCAGAUGUU 1273CAUCUGACAGCUCAGGCUCUU 2776 D-2269 806-824 689-707 AGCCUGAGCUGUCAGAUGGUU1274 CCAUCUGACAGCUCAGGCUUU 2777 D-2270 807-825 690-708GCCUGAGCUGUCAGAUGGCUU 1275 GCCAUCUGACAGCUCAGGCUU 2778 D-2271 808-826691-709 CCUGAGCUGUCAGAUGGCGUU 1276 CGCCAUCUGACAGCUCAGGUU 2779 D-227280-98 80-98 GGCAACCCAUCCCCCACUCUU 1277 GAGUGGGGGAUGGGUUGCCUU 2780 D-2273809-827 692-710 CUGAGCUGUCAGAUGGCGGUU 1278 CCGCCAUCUGACAGCUCAGUU 2781D-2274 810-828 693-711 UGAGCUGUCAGAUGGCGGCUU 1279 GCCGCCAUCUGACAGCUCAUU2782 D-2275 811-829 694-712 GAGCUGUCAGAUGGCGGCGUU 1280CGCCGCCAUCUGACAGCUCUU 2783 D-2276 812-830 695-713 AGCUGUCAGAUGGCGGCGCUU1281 GCGCCGCCAUCUGACAGCUUU 2784 D-2277 813-831 696-714GCUGUCAGAUGGCGGCGCUUU 1282 AGCGCCGCCAUCUGACAGCUU 2785 D-2278 814-832697-715 CUGUCAGAUGGCGGCGCUCUU 1283 GAGCGCCGCCAUCUGACAGUU 2786 D-2279815-833 698-716 UGUCAGAUGGCGGCGCUCCUU 1284 GGAGCGCCGCCAUCUGACAUU 2787D-2280 816-834 699-717 GUCAGAUGGCGGCGCUCCAUU 1285 UGGAGCGCCGCCAUCUGACUU2788 D-2281 817-835 700-718 UCAGAUGGCGGCGCUCCAGUU 1286CUGGAGCGCCGCCAUCUGAUU 2789 D-2282 818-836 701-719 CAGAUGGCGGCGCUCCAGGUU1287 CCUGGAGCGCCGCCAUCUGUU 2790 D-2283 819-837 702-720AGAUGGCGGCGCUCCAGGGUU 1288 CCCUGGAGCGCCGCCAUCUUU 2791 D-2284 81-99 81-99GCAACCCAUCCCCCACUCCUU 1289 GGAGUGGGGGAUGGGUUGCUU 2792 D-2285 820-838703-721 GAUGGCGGCGCUCCAGGGCUU 1290 GCCCUGGAGCGCCGCCAUCUU 2793 D-2286 82-100 82-100 CAACCCAUCCCCCACUCCCUU 1291 GGGAGUGGGGGAUGGGUUGUU 2794D-2287 821-839 704-722 AUGGCGGCGCUCCAGGGCAUU 1292 UGCCCUGGAGCGCCGCCAUUU2795 D-2288 822-840 705-723 UGGCGGCGCUCCAGGGCAAUU 1293UUGCCCUGGAGCGCCGCCAUU 2796 D-2289 823-841 706-724 GGCGGCGCUCCAGGGCAAUUU1294 AUUGCCCUGGAGCGCCGCCUU 2797 D-2290 824-842 707-725GCGGCGCUCCAGGGCAAUGUU 1295 CAUUGCCCUGGAGCGCCGCUU 2798 D-2291 825-843708-726 CGGCGCUCCAGGGCAAUGGUU 1296 CCAUUGCCCUGGAGCGCCGUU 2799 D-2292 8-26  8-26 GGUGCACGGAAGAGUGAGGUU 1297 CCUCACUCUUCCGUGCACCUU 2800 D-2293826-844 709-727 GGCGCUCCAGGGCAAUGGCUU 1298 GCCAUUGCCCUGGAGCGCCUU 2801D-2294 827-845 710-728 GCGCUCCAGGGCAAUGGCUUU 1299 AGCCAUUGCCCUGGAGCGCUU2802 D-2295 828-846 711-729 CGCUCCAGGGCAAUGGCUCUU 1300GAGCCAUUGCCCUGGAGCGUU 2803 D-2296 829-847 712-730 GCUCCAGGGCAAUGGCUCAUU1301 UGAGCCAUUGCCCUGGAGCUU 2804 D-2297 830-848 713-731CUCCAGGGCAAUGGCUCAGUU 1302 CUGAGCCAUUGCCCUGGAGUU 2805 D-2298  83-101 83-101 AACCCAUCCCCCACUCCCAUU 1303 UGGGAGUGGGGGAUGGGUUUU 2806 D-2299831-849 714-732 UCCAGGGCAAUGGCUCAGAUU 1304 UCUGAGCCAUUGCCCUGGAUU 2807D-2300 832-850 715-733 CCAGGGCAAUGGCUCAGAAUU 1305 UUCUGAGCCAUUGCCCUGGUU2808 D-2301 833-851 716-734 CAGGGCAAUGGCUCAGAAAUU 1306UUUCUGAGCCAUUGCCCUGUU 2809 D-2302 834-852 717-735 AGGGCAAUGGCUCAGAAAGUU1307 CUUUCUGAGCCAUUGCCCUUU 2810 D-2303 835-853 718-736GGGCAAUGGCUCAGAAAGGUU 1308 CCUUUCUGAGCCAUUGCCCUU 2811 D-2304 836-854719-737 GGCAAUGGCUCAGAAAGGAUU 1309 UCCUUUCUGAGCCAUUGCCUU 2812 D-2305837-855 720-738 GCAAUGGCUCAGAAAGGACUU 1310 GUCCUUUCUGAGCCAUUGCUU 2813D-2306 838-856 721-739 CAAUGGCUCAGAAAGGACCUU 1311 GGUCCUUUCUGAGCCAUUGUU2814 D-2307 839-857 722-740 AAUGGCUCAGAAAGGACCUUU 1312AGGUCCUUUCUGAGCCAUUUU 2815 D-2308 840-858 723-741 AUGGCUCAGAAAGGACCUGUU1313 CAGGUCCUUUCUGAGCCAUUU 2816 D-2309  84-102  84-102ACCCAUCCCCCACUCCCACUU 1314 GUGGGAGUGGGGGAUGGGUUU 2817 D-2310 841-859724-742 UGGCUCAGAAAGGACCUGCUU 1315 GCAGGUCCUUUCUGAGCCAUU 2818 D-2311842-860 725-743 GGCUCAGAAAGGACCUGCUUU 1316 AGCAGGUCCUUUCUGAGCCUU 2819D-2312 843-861 726-744 GCUCAGAAAGGACCUGCUGUU 1317 CAGCAGGUCCUUUCUGAGCUU2820 D-2313 844-862 727-745 CUCAGAAAGGACCUGCUGCUU 1318GCAGCAGGUCCUUUCUGAGUU 2821 D-2314 845-863 728-746 UCAGAAAGGACCUGCUGCCUU1319 GGCAGCAGGUCCUUUCUGAUU 2822 D-2315 846-864 729-747CAGAAAGGACCUGCUGCCCUU 1320 GGGCAGCAGGUCCUUUCUGUU 2823 D-2316 847-865730-748 AGAAAGGACCUGCUGCCCGUU 1321 CGGGCAGCAGGUCCUUUCUUU 2824 D-2317848-866 731-749 GAAAGGACCUGCUGCCCGGUU 1322 CCGGGCAGCAGGUCCUUUCUU 2825D-2318 849-867 732-750 AAAGGACCUGCUGCCCGGUUU 1323 ACCGGGCAGCAGGUCCUUUUU2826 D-2319 850-868 733-751 AAGGACCUGCUGCCCGGUCUU 1324GACCGGGCAGCAGGUCCUUUU 2827 D-2320  85-103  85-103 CCCAUCCCCCACUCCCACCUU1325 GGUGGGAGUGGGGGAUGGGUU 2828 D-2321 851-869 734-752AGGACCUGCUGCCCGGUCAUU 1326 UGACCGGGCAGCAGGUCCUUU 2829 D-2322 852-870735-753 GGACCUGCUGCCCGGUCAAUU 1327 UUGACCGGGCAGCAGGUCCUU 2830 D-2323853-871 736-754 GACCUGCUGCCCGGUCAACUU 1328 GUUGACCGGGCAGCAGGUCUU 2831D-2324 854-872 737-755 ACCUGCUGCCCGGUCAACUUU 1329 AGUUGACCGGGCAGCAGGUUU2832 D-2325 855-873 738-756 CCUGCUGCCCGGUCAACUGUU 1330CAGUUGACCGGGCAGCAGGUU 2833 D-2326 856-874 739-757 CUGCUGCCCGGUCAACUGGUU1331 CCAGUUGACCGGGCAGCAGUU 2834 D-2327 857-875 740-758UGCUGCCCGGUCAACUGGGUU 1332 CCCAGUUGACCGGGCAGCAUU 2835 D-2328 858-876741-759 GCUGCCCGGUCAACUGGGUUU 1333 ACCCAGUUGACCGGGCAGCUU 2836 D-2329859-877 742-760 CUGCCCGGUCAACUGGGUGUU 1334 CACCCAGUUGACCGGGCAGUU 2837D-2330 860-878 743-761 UGCCCGGUCAACUGGGUGGUU 1335 CCACCCAGUUGACCGGGCAUU2838 D-2331  86-104  86-104 CCAUCCCCCACUCCCACCCUU 1336GGGUGGGAGUGGGGGAUGGUU 2839 D-2332 861-879 744-762 GCCCGGUCAACUGGGUGGAUU1337 UCCACCCAGUUGACCGGGCUU 2840 D-2333 862-880 745-763CCCGGUCAACUGGGUGGAGUU 1338 CUCCACCCAGUUGACCGGGUU 2841 D-2334 863-881746-764 CCGGUCAACUGGGUGGAGCUU 1339 GCUCCACCCAGUUGACCGGUU 2842 D-2335864-882 747-765 CGGUCAACUGGGUGGAGCAUU 1340 UGCUCCACCCAGUUGACCGUU 2843D-2336 865-883 748-766 GGUCAACUGGGUGGAGCACUU 1341 GUGCUCCACCCAGUUGACCUU2844 D-2337 866-884 749-767 GUCAACUGGGUGGAGCACGUU 1342CGUGCUCCACCCAGUUGACUU 2845 D-2338 867-885 750-768 UCAACUGGGUGGAGCACGAUU1343 UCGUGCUCCACCCAGUUGAUU 2846 D-2339 868-886 751-769CAACUGGGUGGAGCACGAGUU 1344 CUCGUGCUCCACCCAGUUGUU 2847 D-2340 869-887752-770 AACUGGGUGGAGCACGAGCUU 1345 GCUCGUGCUCCACCCAGUUUU 2848 D-2341870-888 753-771 ACUGGGUGGAGCACGAGCGUU 1346 CGCUCGUGCUCCACCCAGUUU 2849D-2342  87-105  87-105 CAUCCCCCACUCCCACCCCUU 1347 GGGGUGGGAGUGGGGGAUGUU2850 D-2343 871-889 754-772 CUGGGUGGAGCACGAGCGCUU 1348GCGCUCGUGCUCCACCCAGUU 2851 D-2344 872-890 755-773 UGGGUGGAGCACGAGCGCAUU1349 UGCGCUCGUGCUCCACCCAUU 2852 D-2345 873-891 756-774GGGUGGAGCACGAGCGCAGUU 1350 CUGCGCUCGUGCUCCACCCUU 2853 D-2346 874-892757-775 GGUGGAGCACGAGCGCAGCUU 1351 GCUGCGCUCGUGCUCCACCUU 2854 D-2347875-893 758-776 GUGGAGCACGAGCGCAGCUUU 1352 AGCUGCGCUCGUGCUCCACUU 2855D-2348 876-894 759-777 UGGAGCACGAGCGCAGCUGUU 1353 CAGCUGCGCUCGUGCUCCAUU2856 D-2349 877-895 760-778 GGAGCACGAGCGCAGCUGCUU 1354GCAGCUGCGCUCGUGCUCCUU 2857 D-2350 878-896 761-779 GAGCACGAGCGCAGCUGCUUU1355 AGCAGCUGCGCUCGUGCUCUU 2858 D-2351 879-897 762-780AGCACGAGCGCAGCUGCUAUU 1356 UAGCAGCUGCGCUCGUGCUUU 2859 D-2352 880-898763-781 GCACGAGCGCAGCUGCUACUU 1357 GUAGCAGCUGCGCUCGUGCUU 2860 D-2353 88-106  88-106 AUCCCCCACUCCCACCCCCUU 1358 GGGGGUGGGAGUGGGGGAUUU 2861D-2354 881-899 764-782 CACGAGCGCAGCUGCUACUUU 1359 AGUAGCAGCUGCGCUCGUGUU2862 D-2355 882-900 765-783 ACGAGCGCAGCUGCUACUGUU 1360CAGUAGCAGCUGCGCUCGUUU 2863 D-2356 883-901 766-784 CGAGCGCAGCUGCUACUGGUU1361 CCAGUAGCAGCUGCGCUCGUU 2864 D-2357 884-902 767-785GAGCGCAGCUGCUACUGGUUU 1362 ACCAGUAGCAGCUGCGCUCUU 2865 D-2358 885-903768-786 AGCGCAGCUGCUACUGGUUUU 1363 AACCAGUAGCAGCUGCGCUUU 2866 D-2359886-904 769-787 GCGCAGCUGCUACUGGUUCUU 1364 GAACCAGUAGCAGCUGCGCUU 2867D-2360 887-905 770-788 CGCAGCUGCUACUGGUUCUUU 1365 AGAACCAGUAGCAGCUGCGUU2868 D-2361 888-906 771-789 GCAGCUGCUACUGGUUCUCUU 1366GAGAACCAGUAGCAGCUGCUU 2869 D-2362 889-907 772-790 CAGCUGCUACUGGUUCUCUUU1367 AGAGAACCAGUAGCAGCUGUU 2870 D-2363 890-908 773-791AGCUGCUACUGGUUCUCUCUU 1368 GAGAGAACCAGUAGCAGCUUU 2871 D-2364  89-107 89-107 UCCCCCACUCCCACCCCCAUU 1369 UGGGGGUGGGAGUGGGGGAUU 2872 D-2365891-909 774-792 GCUGCUACUGGUUCUCUCGUU 1370 CGAGAGAACCAGUAGCAGCUU 2873D-2366 892-910 775-793 CUGCUACUGGUUCUCUCGCUU 1371 GCGAGAGAACCAGUAGCAGUU2874 D-2367 893-911 776-794 UGCUACUGGUUCUCUCGCUUU 1372AGCGAGAGAACCAGUAGCAUU 2875 D-2368 894-912 777-795 GCUACUGGUUCUCUCGCUCUU1373 GAGCGAGAGAACCAGUAGCUU 2876 D-2369 895-913 778-796CUACUGGUUCUCUCGCUCCUU 1374 GGAGCGAGAGAACCAGUAGUU 2877 D-2370 896-914779-797 UACUGGUUCUCUCGCUCCGUU 1375 CGGAGCGAGAGAACCAGUAUU 2878 D-2371897-915 780-798 ACUGGUUCUCUCGCUCCGGUU 1376 CCGGAGCGAGAGAACCAGUUU 2879D-2372 898-916 781-799 CUGGUUCUCUCGCUCCGGGUU 1377 CCCGGAGCGAGAGAACCAGUU2880 D-2373 899-917 782-800 UGGUUCUCUCGCUCCGGGAUU 1378UCCCGGAGCGAGAGAACCAUU 2881 D-2374 900-918 783-801 GGUUCUCUCGCUCCGGGAAUU1379 UUCCCGGAGCGAGAGAACCUU 2882 D-2375  90-108  90-108CCCCCACUCCCACCCCCACUU 1380 GUGGGGGUGGGAGUGGGGGUU 2883 D-2376 901-919784-802 GUUCUCUCGCUCCGGGAAGUU 1381 CUUCCCGGAGCGAGAGAACUU 2884 D-2377902-920 785-803 UUCUCUCGCUCCGGGAAGGUU 1382 CCUUCCCGGAGCGAGAGAAUU 2885D-2378 903-921 786-804 UCUCUCGCUCCGGGAAGGCUU 1383 GCCUUCCCGGAGCGAGAGAUU2886 D-2379 904-922 787-805 CUCUCGCUCCGGGAAGGCCUU 1384GGCCUUCCCGGAGCGAGAGUU 2887 D-2380 905-923 788-806 UCUCGCUCCGGGAAGGCCUUU1385 AGGCCUUCCCGGAGCGAGAUU 2888 D-2381 906-924 789-807CUCGCUCCGGGAAGGCCUGUU 1386 CAGGCCUUCCCGGAGCGAGUU 2889 D-2382 907-925790-808 UCGCUCCGGGAAGGCCUGGUU 1387 CCAGGCCUUCCCGGAGCGAUU 2890 D-2383908-926 791-809 CGCUCCGGGAAGGCCUGGGUU 1388 CCCAGGCCUUCCCGGAGCGUU 2891D-2384 909-927 792-810 GCUCCGGGAAGGCCUGGGCUU 1389 GCCCAGGCCUUCCCGGAGCUU2892 D-2385 910-928 793-811 CUCCGGGAAGGCCUGGGCUUU 1390AGCCCAGGCCUUCCCGGAGUU 2893 D-2386  91-109  91-109 CCCCACUCCCACCCCCACAUU1391 UGUGGGGGUGGGAGUGGGGUU 2894 D-2387 911-929 794-812UCCGGGAAGGCCUGGGCUGUU 1392 CAGCCCAGGCCUUCCCGGAUU 2895 D-2388 912-930795-813 CCGGGAAGGCCUGGGCUGAUU 1393 UCAGCCCAGGCCUUCCCGGUU 2896 D-2389913-931 796-814 CGGGAAGGCCUGGGCUGACUU 1394 GUCAGCCCAGGCCUUCCCGUU 2897D-2390 914-932 797-815 GGGAAGGCCUGGGCUGACGUU 1395 CGUCAGCCCAGGCCUUCCCUU2898 D-2391 915-933 798-816 GGAAGGCCUGGGCUGACGCUU 1396GCGUCAGCCCAGGCCUUCCUU 2899 D-2392 916-934 799-817 GAAGGCCUGGGCUGACGCCUU1397 GGCGUCAGCCCAGGCCUUCUU 2900 D-2393 917-935 800-818AAGGCCUGGGCUGACGCCGUU 1398 CGGCGUCAGCCCAGGCCUUUU 2901 D-2394 918-936801-819 AGGCCUGGGCUGACGCCGAUU 1399 UCGGCGUCAGCCCAGGCCUUU 2902 D-2395919-937 802-820 GGCCUGGGCUGACGCCGACUU 1400 GUCGGCGUCAGCCCAGGCCUU 2903D-2396 920-938 803-821 GCCUGGGCUGACGCCGACAUU 1401 UGUCGGCGUCAGCCCAGGCUU2904 D-2397  92-110  92-110 CCCACUCCCACCCCCACACUU 1402GUGUGGGGGUGGGAGUGGGUU 2905 D-2398 921-939 804-822 CCUGGGCUGACGCCGACAAUU1403 UUGUCGGCGUCAGCCCAGGUU 2906 D-2399 922-940 805-823CUGGGCUGACGCCGACAACUU 1404 GUUGUCGGCGUCAGCCCAGUU 2907 D-2400 923-941806-824 UGGGCUGACGCCGACAACUUU 1405 AGUUGUCGGCGUCAGCCCAUU 2908 D-2401924-942 807-825 GGGCUGACGCCGACAACUAUU 1406 UAGUUGUCGGCGUCAGCCCUU 2909D-2402 925-943 808-826 GGCUGACGCCGACAACUACUU 1407 GUAGUUGUCGGCGUCAGCCUU2910 D-2403 926-944 809-827 GCUGACGCCGACAACUACUUU 1408AGUAGUUGUCGGCGUCAGCUU 2911 D-2404  9-27  9-27 GUGCACGGAAGAGUGAGGUUU 1409ACCUCACUCUUCCGUGCACUU 2912 D-2405 927-945 810-828 CUGACGCCGACAACUACUGUU1410 CAGUAGUUGUCGGCGUCAGUU 2913 D-2406 928-946 811-829UGACGCCGACAACUACUGCUU 1411 GCAGUAGUUGUCGGCGUCAUU 2914 D-2407 929-947812-830 GACGCCGACAACUACUGCCUU 1412 GGCAGUAGUUGUCGGCGUCUU 2915 D-2408930-948 813-831 ACGCCGACAACUACUGCCGUU 1413 CGGCAGUAGUUGUCGGCGUUU 2916D-2409  93-111  93-111 CCACUCCCACCCCCACACUUU 1414 AGUGUGGGGGUGGGAGUGGUU2917 D-2410 931-949 814-832 CGCCGACAACUACUGCCGGUU 1415CCGGCAGUAGUUGUCGGCGUU 2918 D-2411 932-950 815-833 GCCGACAACUACUGCCGGCUU1416 GCCGGCAGUAGUUGUCGGCUU 2919 D-2412 933-951 816-834CCGACAACUACUGCCGGCUUU 1417 AGCCGGCAGUAGUUGUCGGUU 2920 D-2413 934-952817-835 CGACAACUACUGCCGGCUGUU 1418 CAGCCGGCAGUAGUUGUCGUU 2921 D-2414935-953 818-836 GACAACUACUGCCGGCUGGUU 1419 CCAGCCGGCAGUAGUUGUCUU 2922D-2415 936-954 819-837 ACAACUACUGCCGGCUGGAUU 1420 UCCAGCCGGCAGUAGUUGUUU2923 D-2416 937-955 820-838 CAACUACUGCCGGCUGGAGUU 1421CUCCAGCCGGCAGUAGUUGUU 2924 D-2417 938-956 821-839 AACUACUGCCGGCUGGAGGUU1422 CCUCCAGCCGGCAGUAGUUUU 2925 D-2418 939-957 822-840ACUACUGCCGGCUGGAGGAUU 1423 UCCUCCAGCCGGCAGUAGUUU 2926 D-2419 940-958823-841 CUACUGCCGGCUGGAGGACUU 1424 GUCCUCCAGCCGGCAGUAGUU 2927 D-2420 94-112  94-112 CACUCCCACCCCCACACUCUU 1425 GAGUGUGGGGGUGGGAGUGUU 2928D-2421 941-959 824-842 UACUGCCGGCUGGAGGACGUU 1426 CGUCCUCCAGCCGGCAGUAUU2929 D-2422 942-960 825-843 ACUGCCGGCUGGAGGACGCUU 1427GCGUCCUCCAGCCGGCAGUUU 2930 D-2423 943-961 826-844 CUGCCGGCUGGAGGACGCGUU1428 CGCGUCCUCCAGCCGGCAGUU 2931 D-2424 944-962 827-845UGCCGGCUGGAGGACGCGCUU 1429 GCGCGUCCUCCAGCCGGCAUU 2932 D-2425 945-963828-846 GCCGGCUGGAGGACGCGCAUU 1430 UGCGCGUCCUCCAGCCGGCUU 2933 D-2426946-964 829-847 CCGGCUGGAGGACGCGCACUU 1431 GUGCGCGUCCUCCAGCCGGUU 2934D-2427 947-965 830-848 CGGCUGGAGGACGCGCACCUU 1432 GGUGCGCGUCCUCCAGCCGUU2935 D-2428 948-966 831-849 GGCUGGAGGACGCGCACCUUU 1433AGGUGCGCGUCCUCCAGCCUU 2936 D-2429 949-967 832-850 GCUGGAGGACGCGCACCUGUU1434 CAGGUGCGCGUCCUCCAGCUU 2937 D-2430 950-968 833-851CUGGAGGACGCGCACCUGGUU 1435 CCAGGUGCGCGUCCUCCAGUU 2938 D-2431  95-113 95-113 ACUCCCACCCCCACACUCCUU 1436 GGAGUGUGGGGGUGGGAGUUU 2939 D-2432951-969 834-852 UGGAGGACGCGCACCUGGUUU 1437 ACCAGGUGCGCGUCCUCCAUU 2940D-2433 952-970 835-853 GGAGGACGCGCACCUGGUGUU 1438 CACCAGGUGCGCGUCCUCCUU2941 D-2434 953-971 836-854 GAGGACGCGCACCUGGUGGUU 1439CCACCAGGUGCGCGUCCUCUU 2942 D-2435 954-972 837-855 AGGACGCGCACCUGGUGGUUU1440 ACCACCAGGUGCGCGUCCUUU 2943 D-2436 955-973 838-856GGACGCGCACCUGGUGGUGUU 1441 CACCACCAGGUGCGCGUCCUU 2944 D-2437 956-974839-857 GACGCGCACCUGGUGGUGGUU 1442 CCACCACCAGGUGCGCGUCUU 2945 D-2438957-975 840-858 ACGCGCACCUGGUGGUGGUUU 1443 ACCACCACCAGGUGCGCGUUU 2946D-2439 958-976 841-859 CGCGCACCUGGUGGUGGUCUU 1444 GACCACCACCAGGUGCGCGUU2947 D-2440 959-977 842-860 GCGCACCUGGUGGUGGUCAUU 1445UGACCACCACCAGGUGCGCUU 2948 D-2441 960-978 843-861 CGCACCUGGUGGUGGUCACUU1446 GUGACCACCACCAGGUGCGUU 2949 D-2442  96-114  96-114CUCCCACCCCCACACUCCCUU 1447 GGGAGUGUGGGGGUGGGAGUU 2950 D-2443 961-979844-862 GCACCUGGUGGUGGUCACGUU 1448 CGUGACCACCACCAGGUGCUU 2951 D-2444962-980 845-863 CACCUGGUGGUGGUCACGUUU 1449 ACGUGACCACCACCAGGUGUU 2952D-2445 963-981 846-864 ACCUGGUGGUGGUCACGUCUU 1450 GACGUGACCACCACCAGGUUU2953 D-2446 964-982 847-865 CCUGGUGGUGGUCACGUCCUU 1451GGACGUGACCACCACCAGGUU 2954 D-2447 965-983 848-866 CUGGUGGUGGUCACGUCCUUU1452 AGGACGUGACCACCACCAGUU 2955 D-2448 966-984 849-867UGGUGGUGGUCACGUCCUGUU 1453 CAGGACGUGACCACCACCAUU 2956 D-2449 967-985850-868 GGUGGUGGUCACGUCCUGGUU 1454 CCAGGACGUGACCACCACCUU 2957 D-2450968-986 851-869 GUGGUGGUCACGUCCUGGGUU 1455 CCCAGGACGUGACCACCACUU 2958D-2451 969-987 852-870 UGGUGGUCACGUCCUGGGAUU 1456 UCCCAGGACGUGACCACCAUU2959 D-2452 970-988 853-871 GGUGGUCACGUCCUGGGAGUU 1457CUCCCAGGACGUGACCACCUU 2960 D-2453  97-115  97-115 UCCCACCCCCACACUCCCCUU1458 GGGGAGUGUGGGGGUGGGAUU 2961 D-2454 971-989 854-872GUGGUCACGUCCUGGGAGGUU 1459 CCUCCCAGGACGUGACCACUU 2962 D-2455 972-990855-873 UGGUCACGUCCUGGGAGGAUU 1460 UCCUCCCAGGACGUGACCAUU 2963 D-2456973-991 856-874 GGUCACGUCCUGGGAGGAGUU 1461 CUCCUCCCAGGACGUGACCUU 2964D-2457 974-992 857-875 GUCACGUCCUGGGAGGAGCUU 1462 GCUCCUCCCAGGACGUGACUU2965 D-2458 975-993 858-876 UCACGUCCUGGGAGGAGCAUU 1463UGCUCCUCCCAGGACGUGAUU 2966 D-2459 976-994 859-877 CACGUCCUGGGAGGAGCAGUU1464 CUGCUCCUCCCAGGACGUGUU 2967 D-2460 977-995 860-878ACGUCCUGGGAGGAGCAGAUU 1465 UCUGCUCCUCCCAGGACGUUU 2968 D-2461 978-996861-879 CGUCCUGGGAGGAGCAGAAUU 1466 UUCUGCUCCUCCCAGGACGUU 2969 D-2462979-997 862-880 GUCCUGGGAGGAGCAGAAAUU 1467 UUUCUGCUCCUCCCAGGACUU 2970D-2463 980-998 863-881 UCCUGGGAGGAGCAGAAAUUU 1468 AUUUCUGCUCCUCCCAGGAUU2971 D-2464  98-116  98-116 CCCACCCCCACACUCCCCUUU 1469AGGGGAGUGUGGGGGUGGGUU 2972 D-2465 981-999 864-882 CCUGGGAGGAGCAGAAAUUUU1470 AAUUUCUGCUCCUCCCAGGUU 2973 D-2466  982-1000 865-883CUGGGAGGAGCAGAAAUUUUU 1471 AAAUUUCUGCUCCUCCCAGUU 2974 D-2467  983-1001866-884 UGGGAGGAGCAGAAAUUUGUU 1472 CAAAUUUCUGCUCCUCCCAUU 2975 D-2468 984-1002 867-885 GGGAGGAGCAGAAAUUUGUUU 1473 ACAAAUUUCUGCUCCUCCCUU 2976D-2469  985-1003 868-886 GGAGGAGCAGAAAUUUGUCUU 1474GACAAAUUUCUGCUCCUCCUU 2977 D-2470  986-1004 869-887GAGGAGCAGAAAUUUGUCCUU 1475 GGACAAAUUUCUGCUCCUCUU 2978 D-2471  987-1005870-888 AGGAGCAGAAAUUUGUCCAUU 1476 UGGACAAAUUUCUGCUCCUUU 2979 D-2472 988-1006 871-889 GGAGCAGAAAUUUGUCCAGUU 1477 CUGGACAAAUUUCUGCUCCUU 2980D-2473  989-1007 872-890 GAGCAGAAAUUUGUCCAGCUU 1478GCUGGACAAAUUUCUGCUCUU 2981 D-2474  990-1008 873-891AGCAGAAAUUUGUCCAGCAUU 1479 UGCUGGACAAAUUUCUGCUUU 2982 D-2475  991-1009874-892 GCAGAAAUUUGUCCAGCACUU 1480 GUGCUGGACAAAUUUCUGCUU 2983 D-2476 99-117  99-117 CCACCCCCACACUCCCCUAUU 1481 UAGGGGAGUGUGGGGGUGGUU 2984D-2477  992-1010 875-893 CAGAAAUUUGUCCAGCACCUU 1482GGUGCUGGACAAAUUUCUGUU 2985 D-2478  993-1011 876-894AGAAAUUUGUCCAGCACCAUU 1483 UGGUGCUGGACAAAUUUCUUU 2986 D-2479  994-1012877-895 GAAAUUUGUCCAGCACCACUU 1484 GUGGUGCUGGACAAAUUUCUU 2987 D-2480 995-1013 878-896 AAAUUUGUCCAGCACCACAUU 1485 UGUGGUGCUGGACAAAUUUUU 2988D-2481  996-1014 879-897 AAUUUGUCCAGCACCACAUUU 1486AUGUGGUGCUGGACAAAUUUU 2989 D-2482  997-1015 880-898AUUUGUCCAGCACCACAUAUU 1487 UAUGUGGUGCUGGACAAAUUU 2990 D-2483  998-1016881-899 UUUGUCCAGCACCACAUAGUU 1488 CUAUGUGGUGCUGGACAAAUU 2991 D-2484 999-1017 882-900 UUGUCCAGCACCACAUAGGUU 1489 CCUAUGUGGUGCUGGACAAUU 2992D-2485 — 453-471 ACCAUCAGCUCAGAAAAGAUU 1490 UCUUUUCUGAGCUGAUGGUUU 2993D-2486 — 454-472 CCAUCAGCUCAGAAAAGACUU 1491 GUCUUUUCUGAGCUGAUGGUU 2994D-2487 — 455-473 CAUCAGCUCAGAAAAGACUUU 1492 AGUCUUUUCUGAGCUGAUGUU 2995D-2488 — 456-474 AUCAGCUCAGAAAAGACUCUU 1493 GAGUCUUUUCUGAGCUGAUUU 2996D-2489 — 457-475 UCAGCUCAGAAAAGACUCCUU 1494 GGAGUCUUUUCUGAGCUGAUU 2997D-2490 — 458-476 CAGCUCAGAAAAGACUCCCUU 1495 GGGAGUCUUUUCUGAGCUGUU 2998D-2491 — 459-477 AGCUCAGAAAAGACUCCCAUU 1496 UGGGAGUCUUUUCUGAGCUUU 2999D-2492 — 460-478 GCUCAGAAAAGACUCCCAGUU 1497 CUGGGAGUCUUUUCUGAGCUU 3000D-2493 — 461-479 CUCAGAAAAGACUCCCAGCUU 1498 GCUGGGAGUCUUUUCUGAGUU 3001D-2494 — 462-480 UCAGAAAAGACUCCCAGCUUU 1499 AGCUGGGAGUCUUUUCUGAUU 3002D-2495 — 463-481 CAGAAAAGACUCCCAGCUGUU 1500 CAGCUGGGAGUCUUUUCUGUU 3003D-2496 — 464-482 AGAAAAGACUCCCAGCUGCUU 1501 GCAGCUGGGAGUCUUUUCUUU 3004D-2497 — 465-483 GAAAAGACUCCCAGCUGCAUU 1502 UGCAGCUGGGAGUCUUUUCUU 3005D-2498 — 466-484 AAAAGACUCCCAGCUGCAGUU 1503 CUGCAGCUGGGAGUCUUUUUU 3006D-2499 — 467-485 AAAGACUCCCAGCUGCAGGUU 1504 CCUGCAGCUGGGAGUCUUUUU 3007D-2500 — 468-486 AAGACUCCCAGCUGCAGGAUU 1505 UCCUGCAGCUGGGAGUCUUUU 3008D-2501 — 469-487 AGACUCCCAGCUGCAGGAGUU 1506 CUCCUGCAGCUGGGAGUCUUU 3009D-2502 — 470-488 GACUCCCAGCUGCAGGAGGUU 1507 CCUCCUGCAGCUGGGAGUCUU 3010

Example 2. Efficacy of ASGR1 siRNA Molecules In Vitro

The siRNA molecules in Tier 1 and Tier 2 screening sets were synthesizedwithout chemical modifications. Each siRNA molecule was comprised of a21 nucleotide sense strand and 21 nucleotide antisense strand thathybridized to form a duplex region of 19 base pairs with a 2 nucleotideoverhang at the 3′ end of each strand. The efficacy of each of the siRNAmolecules in reducing ASGR1 expression was assessed using a 384-wellformat in vitro immunoassay, which quantifies levels of ASGR1 protein onthe cell surface of Hep3B or HepG2 cells.

Transfection complexes of the siRNA molecules and RNAiMax transfectionreagent (Life Technologies) in EMEM media (ATCC 30-2003) were preparedin 384-well plates in accordance with manufacturer's recommendations.Human hepatocellular carcinoma Hep3B (ATCC HB-8064) or HepG2 (ATCCHB-8065) cells in EMEM media supplemented with 10% fetal bovine serumand 1% antibiotic/antimycotic were added to each well. Cells wereincubated for 4 days at 37° C. and 5% CO₂. Four days after siRNAtransfection, cells were fixed in formaldehyde, blocked with bovineserum albumin, and subsequently stained with an anti-ASGR1 primaryantibody (Amgen clone 7E11, light and heavy chain sequences providedbelow (SEQ ID NOs: 3 and 4)) for either 1 hour at room temperature orovernight at 4° C. Plates were washed three times with phosphatebuffered saline (PBS). Cells were then incubated in the dark for 45minutes at room temperature with Alexa488-conjugated anti-human IgGsecondary antibody and nuclear stain DRAQ5 (ThermoFisher #62251), whichwas included to assess cell number. Following three PBS washes, theplates were imaged on an Opera Phenix high-content screening system(PerkinElmer) using the 488 and 640 channels to measure anti-ASGR1antibody staining and nuclear staining, respectively. Data was analyzedusing Columbus image analysis software and GeneData Screener software toquantify several measures of ASGR1 protein levels, cell count, and cellmorphology on a per cell and per well basis.

Anti-ASGR1 primary antibody light chain amino acid sequence:

(SEQ ID NO: 3) DIQMTQSPSSLSASVGDRVTIACRASQNIISYLNWYQQKPGKAPKFLIYTASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAIYYCQQTYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Anti-ASGR1 primary antibody heavy chain amino acid sequence:

(SEQ ID NO: 4) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAIIWHDGSNKYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCARDLSMGGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Activity of each siRNA molecule was measured using a “Normalized Alexa488 Mean Intensity” readout, which quantifies ASGR1 protein expressionbased on cell population analysis relative to ASGR1 expression incontrol cell populations. Cells transfected with non-targeting siRNAduplexes (i.e. siRNAs that do not have a 100% sequence match to anyhuman gene sequences) were used as controls on each plate and a “centralreference value” was calculated from these control cells. Specifically,the “central reference value” was the median value of the Alexa 488intensity of the multiple wells containing cells transfected withnon-targeting siRNAs. The “Normalized Alexa 488 Mean Intensity” wascalculated for each well as follows. Nuclei and cytoplasm were segmentedusing the DRAQ5 counterstain. The mean Alexa488 fluorescence intensityfor each cell (i.e. the entire cellular region, including cytoplasm andnucleus) was measured. Individual cell values were averaged to produce amean Alexa 488 intensity value for each well. This mean intensity valuewas then normalized to the central reference value to arrive at the“Normalized Alexa 488 Mean Intensity” value, which represents an ASGR1expression measurement as a percent of control. Thus, negative“Normalized Alexa 488 Mean Intensity” values represent a reduced ASGR1expression relative to control cells. Cell counts as assessed by theDRAQ5 stain were also normalized to cell counts for control cells andthus, represent a cell viability measurement as a percent of control.

The Tier 1 siRNA molecules were tested in duplicate at three differentconcentrations (0.3 nM, 1.25 nM, and 5 nM) in the in vitro immunoassayin both Hep3B cells and HepG2 cells. The reduction in ASGR1 cell surfaceexpression relative to expression in cells transfected withnon-targeting siRNAs in Hep3B cells for each siRNA molecule is shown inTable 2 below. Cell count measurements are also provided. For clarity,the data for the lowest concentration (0.3 nM) and the data for theHepG2 cells are not shown. The data shown in Table 2 are the resultsfrom two independent transfections of each siRNA (e.g. Run 1 and Run 2).

TABLE 2 In vitro efficacy of Tier 1 ASGR1 siRNA molecules in immunoassayscreen Run 1 Run 2 Normalized Normalized Normalized Normalized Alexa 488Alexa 488 Alexa 488 Alexa 488 Cell Mean Cell Mean Cell Mean Cell MeanDuplex count Intensity count Intensity count Intensity count IntensityNo. 5 nM 5 nM 1.25 nM 1.25 nM 5 nM 5 nM 1.25 nM 1.25 nM D-1011 16.51−51.78 0.13 −14.40 25.37 −51.83 3.76 −28.05 D-1018 −17.30 −55.17 2.29−16.02 −34.78 −52.13 30.89 −7.73 D-1024 −29.00 −18.51 −0.82 12.30 1.9714.82 6.60 4.61 D-1036 −46.65 10.97 −10.56 7.07 −18.48 71.68 1.10 12.95D-1055 10.56 −13.14 −1.08 −9.93 7.75 −24.90 11.92 −7.81 D-1063 9.47−18.69 −2.54 −11.47 −25.16 −50.11 −10.17 −18.86 D-1069 −20.87 −41.89−4.27 3.93 −11.39 −28.60 12.28 7.84 D-1082 17.70 −48.78 −8.32 −14.13−0.15 −55.38 −5.13 −50.19 D-1089 −35.75 −60.09 −12.25 −36.42 −25.97−57.46 −7.24 −51.34 D-1091 −18.99 −31.14 −13.76 5.02 −27.59 −20.03 3.02−6.61 D-1098 6.89 −54.37 −2.80 −34.02 −14.23 −59.04 −11.92 −53.04 D-113510.36 −7.69 −15.39 −4.17 24.96 −24.64 3.57 −5.25 D-1147 20.97 −3.11 4.361.69 11.90 5.13 13.93 4.34 D-1151 −4.71 −14.13 −15.57 −17.87 −12.91−34.11 −1.19 −14.79 D-1158 26.52 −10.17 23.24 8.03 28.20 −6.52 18.06−2.88 D-1162 −40.01 −55.60 −22.04 −36.60 −35.29 −52.50 −14.02 −41.72D-1165 −3.62 −54.73 −7.63 −23.22 −0.86 −51.86 15.67 −18.51 D-1166 −3.32−41.79 −11.34 −13.26 −0.25 −14.66 −4.12 −15.13 D-1167 −53.20 −59.29−6.34 −16.20 −34.68 −52.65 2.38 −24.06 D-1168 −31.18 −46.37 −1.51 −9.31−4.10 −34.17 −15.22 −20.42 D-1170 −12.35 −53.90 −3.32 −42.65 −23.85−56.63 5.59 −32.93 D-1171 3.82 −31.95 −14.62 −23.16 −12.00 −49.39 9.90−28.40 D-1172 −45.07 −58.26 −1.94 3.83 −40.15 −56.40 −18.42 −45.04D-1173 −25.53 −57.81 5.39 −8.67 −12.00 −58.53 −24.75 −56.05 D-1174 11.06−32.05 3.06 −15.60 −18.38 −54.41 −13.38 −40.99 D-1175 −35.25 −47.56−9.96 −1.38 −31.75 −44.76 −24.47 −34.12 D-1176 −21.07 −48.99 −12.46−22.28 −55.65 −30.89 −4.67 −34.03 D-1206 −20.58 −58.56 −5.65 −42.60−4.41 −58.69 13.47 −45.33 D-1234 0.74 −45.36 −6.51 4.14 13.42 −37.24−0.46 −19.91 D-1235 −11.85 −55.44 −20.74 −53.47 −36.41 −59.01 −20.26−54.37 D-1237 −20.08 −20.98 5.99 19.18 4.30 −22.26 0.55 −4.37 D-124618.79 −44.97 −0.22 −40.27 −18.38 −57.87 7.97 −44.91 D-1250 −6.10 −51.900.82 2.19 0.15 −58.42 10.08 −49.16 D-1257 −10.36 −55.00 5.39 16.17−20.30 −53.64 −15.58 −41.86 D-1285 −15.32 −50.05 8.93 9.74 −18.08 −56.70−12.37 −45.36 D-1286 −30.00 −55.94 −0.69 −28.77 −11.19 −48.78 −2.70−42.37 D-1288 −26.13 −60.85 3.32 −3.82 −9.16 −56.60 8.07 −36.50 D-1289−11.95 −48.94 −13.41 −9.24 −3.49 −53.30 −9.72 −31.07 D-1291 −21.57−62.92 −2.37 4.10 10.28 −57.93 −5.22 −46.85 D-1293 7.88 −59.68 −4.18−17.53 −1.47 −60.64 1.56 −48.52 D-1294 −13.93 −61.42 9.01 −8.13 4.20−58.50 7.79 −49.89 D-1296 32.37 −35.87 −3.58 −26.73 21.72 −42.10 28.96−8.80 D-1299 9.97 −15.14 5.99 −4.68 14.33 −12.03 −4.77 −16.17 D-130142.98 8.71 10.05 11.59 18.48 −7.91 1.65 0.99 D-1302 8.78 −15.98 11.084.83 4.61 −11.01 −0.73 −2.26 D-1303 18.39 8.99 15.05 3.34 34.38 16.394.95 0.93 D-1335 −4.41 −57.42 −7.81 −12.59 −2.78 −53.97 −5.32 −38.92D-1340 15.42 −56.65 12.63 −47.35 18.08 −56.73 11.55 −47.26 D-1342 −1.34−55.68 1.60 −30.81 −14.13 −55.74 24.66 −34.02 D-1350 12.25 −46.29 16.43−17.81 8.86 −45.60 11.73 −15.78 D-1364 −36.14 −58.31 −7.81 −45.74 −31.95−57.24 −2.47 −42.87 D-1365 −45.46 −59.21 1.08 −39.04 −54.43 −55.49 −9.26−47.65 D-1367 −2.23 −49.60 27.47 −0.24 0.35 −55.94 15.49 −40.01 D-137329.80 −55.97 22.64 −34.90 −9.37 −57.64 15.40 −50.30 D-1376 9.37 −54.034.61 −6.55 −18.48 −58.89 23.74 −40.26 D-1387 −15.62 −60.74 12.03 −24.43−22.84 −59.13 −0.46 −55.15 D-1389 37.33 −60.30 −1.68 −58.97 −6.03 −59.856.51 −56.49 D-1390 −7.09 −62.69 1.94 −19.96 18.08 −60.28 11.92 −58.45D-1397 −17.90 −62.94 10.13 −56.73 25.06 −60.77 4.12 −56.43 D-1398 −7.09−61.91 9.27 −39.40 −6.43 −56.41 27.77 −34.30 D-1399 −33.81 −62.45 12.59−17.06 2.08 −59.75 11.14 −50.83 D-1400 46.65 −59.41 13.50 −36.58 16.15−59.11 11.92 −48.21 D-1403 24.74 −0.88 20.40 4.38 −19.29 −29.75 11.274.71 D-1405 −7.78 −10.82 8.24 −5.29 4.30 −1.20 −4.03 −5.98 D-1406 −3.92−9.59 −1.85 −8.89 −5.01 −9.40 23.74 15.19 D-1407 18.69 −3.89 −7.29−12.59 −4.30 −19.11 9.90 −6.78 D-1408 8.73 −49.20 −12.98 −42.77 −1.47−53.33 12.19 −35.80 D-1426 −6.79 −61.40 −12.46 −51.50 −4.30 −59.00−19.34 −56.51 D-1431 5.30 −58.85 −8.50 −53.37 −3.19 −57.76 0.18 −47.13D-1432 −16.11 −61.98 −2.72 −45.35 −22.53 −58.60 3.76 −47.92 D-1438 7.09−61.80 23.59 −55.97 3.49 −58.39 21.45 −54.63 D-1443 −10.76 −62.32 2.98−59.31 3.80 −59.01 8.98 −54.86 D-1452 −0.79 −56.32 14.27 −45.83 −31.70−56.68 10.91 −52.19 D-1453 12.05 −56.42 31.87 −26.99 −32.86 −57.12 13.47−56.95 D-1454 −25.83 −60.61 2.63 −24.99 −23.95 −59.91 9.62 −55.41 D-145519.58 −55.42 −6.34 −27.92 8.46 −58.89 0.27 −51.97 D-1472 29.80 −55.21−2.89 −22.04 −13.42 −58.75 21.91 −52.79 D-1478 −3.12 −60.56 7.03 −3.284.81 −58.19 12.01 −43.07 D-1479 20.38 −59.07 −15.14 −22.23 −7.95 −58.4110.08 −42.61 D-1484 −6.30 −62.62 9.87 −39.24 13.01 −58.78 3.76 −51.54D-1494 −13.73 −63.95 6.34 −41.07 −11.19 −60.62 22.64 −59.92 D-1495 8.28−62.15 4.70 −27.22 18.89 −60.38 20.16 −55.68 D-1497 26.13 −59.97 4.96−58.89 0.05 −60.05 14.12 −58.30 D-1502 14.97 −45.58 1.94 −38.33 −4.76−54.72 5.77 −38.10 D-1505 −8.28 −59.54 −12.25 −17.83 −14.08 −59.50 3.02−36.43 D-1506 8.18 −57.99 20.91 −9.71 −2.48 −58.59 2.11 −41.88 D-1511−0.74 −15.67 −2.63 −10.15 1.16 −14.97 13.84 −0.23 D-1516 12.05 −4.94−1.08 0.63 4.51 1.35 −1.19 −17.05 D-1518 −4.21 −26.51 −13.58 −8.51 −4.00−3.46 13.57 −3.88 D-1521 18.29 −23.36 6.68 1.40 20.81 15.51 −3.85 −13.25D-1536 16.31 −15.47 1.25 5.23 27.70 −11.54 6.14 0.40 D-1554 13.24 −13.40−4.70 0.95 6.63 −32.03 3.30 −0.92 D-1556 −26.52 −41.44 3.49 6.92 −3.19−13.71 −1.47 −10.81 D-1572 26.62 −24.09 20.31 5.52 5.62 −1.00 24.0111.58 D-1578 −4.71 −44.74 −12.89 −14.92 2.78 −23.50 5.22 −5.92 D-1581−23.05 −27.51 −16.17 −14.43 −22.53 −12.89 −2.38 −15.63 D-1623 −22.46−30.62 −11.69 −6.55 −25.27 −32.19 13.47 4.71 D-1627 12.25 −23.19 −4.96−29.97 −10.89 −38.16 1.56 −25.15 D-1653 11.06 −32.19 9.44 0.09 −4.71−32.15 19.52 0.81 D-1656 5.60 −38.39 1.51 −22.78 −21.42 −49.42 3.94−21.36 D-1678 −32.77 −50.41 −16.52 −23.47 −15.04 −44.98 5.50 −6.64D-1683 11.65 −32.74 4.53 −1.23 4.81 −26.86 13.75 −6.80 D-1686 2.63−31.87 −13.15 −4.49 13.42 −13.30 23.37 −3.19 D-1687 −1.54 −38.12 −0.56−8.62 −17.37 −40.05 21.08 −7.89 D-1694 −15.82 −54.86 8.58 −24.98 −29.82−48.50 14.02 −28.37 D-1708 −17.80 −55.25 −2.03 −31.77 −34.99 −52.79 9.62−30.91 D-1709 −8.28 −48.76 10.65 −2.99 9.06 −33.13 33.36 −2.17 D-1713−6.40 −19.58 −18.76 10.36 −7.85 −17.32 10.91 10.44 D-1716 14.03 −45.6022.38 −5.99 18.78 −49.38 8.71 −45.19 D-1719 −17.50 −53.30 8.75 −6.89−29.92 −56.30 17.69 −20.17 D-1722 22.06 −49.25 6.94 −6.97 2.99 −53.3216.96 −42.54 D-1734 27.32 −44.60 14.19 0.24 −7.04 −56.49 1.10 −44.07D-1741 −2.63 −45.97 2.80 −2.87 −32.15 −55.22 16.68 −18.26 D-1748 7.09−50.05 11.51 −16.11 −34.28 −57.38 8.98 −36.62 D-1750 39.91 −43.78 12.8115.45 30.63 −43.98 24.38 −12.48 D-1752 1.54 −56.17 −3.23 −14.35 15.54−53.71 10.91 −34.83 D-1773 −11.25 −61.04 7.72 −10.35 −19.80 −58.31 20.53−50.30 D-1777 −11.16 −53.95 −1.51 −27.84 −0.05 −50.07 14.57 −28.05D-1792 4.51 −10.06 7.98 2.31 31.44 −0.79 15.67 −0.52 D-1798 −29.00−58.63 6.17 −49.28 −32.05 −58.49 8.71 −54.61 D-1801 19.58 −52.66 −11.00−46.00 −13.62 −58.81 12.65 −45.62 D-1807 14.33 −36.45 −3.41 −8.24 1.57−47.37 6.87 −33.08 D-1808 21.77 −50.54 5.48 −1.08 −25.16 −58.30 14.12−44.68 D-1811 26.92 −48.75 −9.27 −37.36 10.89 −59.48 −2.38 −43.87 D-181315.82 −59.88 5.30 −5.72 48.86 −55.18 12.83 −51.46 D-1814 9.47 −11.40−4.10 5.65 −6.53 −25.64 6.97 6.28 D-1815 20.18 −57.38 0.39 −46.20 3.80−59.35 8.07 −49.05 D-1819 −14.33 −53.70 −4.53 −25.39 −21.62 −29.60 −2.02−34.84 D-1824 8.58 −61.67 2.03 −52.48 −1.97 −60.35 0.73 −48.34 D-1826−1.14 −59.23 3.75 −55.52 −1.47 −59.41 0.82 −56.95 D-1832 22.06 −57.11−7.63 −32.20 −8.25 −59.70 −12.01 −58.04 D-1840 18.89 −48.93 12.29 −24.02−14.33 −57.91 8.52 −41.61 D-1842 8.18 −57.58 −2.63 −11.59 −22.63 −59.429.99 −52.24 D-1848 3.12 1.44 −12.81 −3.07 −24.35 −34.25 −14.57 −5.02D-1874 −1.14 −8.57 −3.88 1.72 −24.25 0.91 −13.79 −3.21 D-1876 12.10−54.54 −0.78 −5.14 17.01 −53.11 5.18 −29.31 D-1929 −4.51 −53.02 −8.50−37.65 −9.97 −56.26 15.86 −35.66 D-1975 −1.83 −60.71 −8.06 −22.41 24.86−59.22 6.05 −45.07 D-1981 4.81 −59.13 −3.67 −21.74 19.29 −57.31 12.56−49.72 D-1983 25.63 −60.82 −6.94 −53.04 18.28 −59.21 13.38 −50.18 D-1987−0.25 −51.50 −17.12 −5.68 −7.34 −56.91 −0.37 −49.23 D-1989 25.93 −59.04−0.22 −56.06 19.70 −59.14 −0.37 −57.37 D-1994 −30.59 −40.01 16.08 −2.53−26.18 −40.53 −20.81 −34.18 D-1999 −5.30 −59.22 −0.22 −28.91 −3.90−54.46 −11.00 −58.90 D-2000 −6.40 −52.51 −10.22 −41.18 −13.92 −56.51−6.78 −47.04 D-2006 −6.10 −56.48 −11.00 −34.67 −23.75 −59.18 −17.14−47.29 D-2008 1.54 −54.28 3.67 −27.83 −17.27 −59.63 −12.37 −44.60 D-2010−6.69 −53.88 −4.27 −9.69 −11.39 −58.47 4.58 −44.37 D-2021 −10.96 −53.49−18.84 −40.95 −1.27 −53.90 13.38 −33.91 D-2045 −26.82 −58.06 4.87 −22.24−22.13 −56.67 −14.48 −39.83 D-2050 −5.01 −32.58 −17.81 −23.38 −13.52−49.82 −11.82 −31.36 D-2054 14.33 −46.36 −18.50 −38.37 −11.90 −53.122.20 −26.56 D-2056 6.89 −56.26 −21.26 −48.12 −5.22 −60.11 0.09 −45.21D-2059 −25.53 −60.64 −4.01 −12.60 −29.52 −59.15 −7.79 −56.33 D-2064−42.29 −55.85 −2.37 −0.05 −37.01 −59.20 2.57 −49.72 D-2066 −27.12 −62.040.56 −21.81 −19.39 −58.62 −9.17 −56.56 D-2069 −2.63 −61.10 0.04 −43.1714.13 −57.57 2.84 −43.86 D-2081 −16.66 −47.70 −3.41 −8.40 17.62 −54.093.16 −27.64 D-2095 −4.51 −59.69 −7.72 −31.26 −3.90 −56.37 13.84 −27.54D-2107 −40.01 −57.62 0.73 −1.33 −19.70 −48.12 −0.64 −30.71 D-2115 −39.12−41.56 −26.35 −31.43 −40.86 −31.71 −3.21 −20.71 D-2122 −42.09 −54.90−19.62 −36.56 −37.22 −56.76 0.82 −33.96 D-2124 −8.08 −46.96 −14.19 −0.08−17.37 −47.88 −21.54 −22.67 D-2130 −5.40 −22.16 3.41 11.97 −17.57 −33.56−18.52 −18.45 D-2133 −25.93 −46.79 −19.97 −5.63 −36.51 −50.48 8.16−10.25 D-2134 −39.81 −61.69 −16.26 −31.95 −22.43 −59.20 −1.74 −34.15D-2136 −11.80 −42.20 −6.34 −4.68 −21.87 −56.73 −6.32 −21.07 D-2137−30.59 −61.11 −10.74 −24.82 5.62 −49.50 17.42 −17.59 D-2142 −26.82−56.29 14.79 3.81 2.68 −46.12 6.51 −21.91 D-2143 −35.25 −56.55 −11.08−41.09 −21.32 −58.35 13.84 −32.12 D-2172 7.29 −29.17 −1.34 12.93 −6.03−34.12 13.11 11.63 D-2174 2.03 −51.87 4.79 4.73 6.84 −47.31 7.97 −30.31D-2192 14.43 −41.72 15.48 −11.07 24.96 −44.47 21.91 −24.19 D-2196 1.64−52.44 14.27 −0.40 −34.38 −55.66 −0.82 −47.45 D-2199 26.82 −56.29 −4.01−2.81 20.71 −58.07 12.37 −38.76 D-2200 5.01 −38.18 8.50 9.77 12.51−44.01 18.42 −25.19 D-2202 5.85 −5.08 −2.24 1.89 −1.47 6.11 1.37 −0.43D-2210 −20.87 −58.61 2.29 −24.34 −1.57 −44.74 15.58 −23.79 D-2224 13.34−54.07 14.70 −17.77 39.54 −51.64 2.57 −36.75 D-2230 −23.95 −40.70 −2.54−5.35 −10.68 −6.76 20.26 15.29 D-2236 9.17 −55.50 −4.44 −35.30 4.51−46.56 −3.57 −27.54 D-2238 16.01 −48.75 −9.79 −29.17 21.42 −42.21 6.14−28.27 D-2266 8.08 −14.56 3.84 −18.65 6.73 −28.22 6.51 −12.90 D-22679.27 0.67 −1.60 3.64 −41.87 −43.95 14.85 15.50 D-2294 25.63 −7.76 14.016.68 −8.66 −31.02 17.78 4.99 D-2296 16.81 −28.11 −1.94 −17.85 −13.22−41.06 17.32 −11.82 D-2300 −26.13 −57.30 8.32 0.46 −45.22 −53.30 3.94−29.90 D-2301 −15.72 −38.46 22.38 5.62 −9.87 −38.50 −9.07 −20.00 D-230414.53 −53.06 4.79 −16.86 −20.91 −58.98 15.03 −42.20 D-2311 −60.73 −63.051.25 −54.94 −59.90 −59.83 6.78 −51.22 D-2322 9.07 −53.47 −1.60 −28.61−3.70 −53.94 5.32 −34.29 D-2335 2.73 −59.25 7.46 −44.73 −3.09 −57.832.02 −37.63 D-2354 3.12 −52.45 16.77 7.77 7.04 −49.64 15.31 −39.67D-2357 46.55 −51.86 14.45 −45.90 3.39 −59.31 3.85 −56.37 D-2360 22.46−57.37 24.71 −13.55 19.49 −56.45 19.80 −34.93 D-2361 11.16 −50.62 10.31−17.97 42.58 −57.92 12.37 −49.42 D-2362 24.54 −24.39 5.22 3.25 10.08−36.37 17.14 −0.36 D-2374 32.97 −24.51 −9.10 1.81 13.01 −36.58 9.07−25.51 D-2388 −18.59 −43.66 12.38 0.51 −10.58 −29.34 30.98 10.51 D-2399−39.22 −56.10 −4.18 −28.95 −68.10 −59.66 −39.60 −51.94 D-2401 −30.49−59.59 −1.25 −11.64 −40.30 −57.59 10.04 −34.66 D-2403 −8.97 −61.28 2.20−9.13 −14.63 −57.17 38.59 −13.48 D-2412 15.62 6.15 12.89 11.91 2.18−12.33 12.10 −4.72 D-2461 −19.78 −56.95 −6.34 −54.93 −35.49 −57.76 −7.52−49.34 D-2462 18.79 −21.80 11.25 −2.95 −32.35 −52.99 −2.84 −24.30 D-246513.83 −55.08 7.89 −7.64 1.97 −58.56 20.90 −43.55 D-2466 28.90 −49.02−7.29 −8.54 −17.47 −57.63 17.05 −43.13 D-2468 9.17 −52.16 −5.74 −14.2543.70 −57.19 12.47 −48.60 D-2475 −28.90 −54.23 −7.12 −5.24 −47.75 −56.375.04 −38.94 D-2481 12.94 −55.66 3.23 −12.64 3.70 −54.71 18.42 −28.59D-2487 17.50 −10.84 −4.01 −0.05 9.37 −7.38 14.12 1.83

Of the 211 unmodified siRNA molecules screened in Tier 1, at the 5 nMconcentration, about 168 siRNA molecules reduced ASGR1 cell surfaceexpression relative to control cells by at least 30%, about 119 siRNAmolecules reduced ASGR1 cell surface expression relative to controlcells by at least 50%, and about 30 siRNA molecules reduced ASGR1 cellsurface expression relative to control cells by at least 60%. Some ofthe siRNA molecules exhibited no or marginal reduction in ASGR1 cellsurface expression relative to control cells.

A subset of the siRNA molecules from this initial screen of the Tier 1molecules were selected for further testing in a 10-point dose responseformat (0.004 nM to 83 nM) in the in vitro anti-ASGR1 immunoassay inHep3B cells. IC50 values for each of these 55 siRNA molecules werecalculated from the dose-response curves and are shown in Table 3 below.Data from two independent transfections of each siRNA molecule (Run 1and Run 2) are shown. At least 18 of the siRNA molecules had IC50 valuesof about 0.30 nM or less. Compounds D-1983, D-1098, D-1438, D-1246, andD-1494 were among the most potent with average IC50 values less than0.15 nM.

TABLE 3 IC50 values determined by immunoassay for select ASGR1 siRNAmolecules Duplex No. Run 1 IC50 (nM) Run 2 IC50 (nM)D-1055 >83.33 >83.33 D-1082 0.72 1.10 D-1089 0.15 0.24 D-1098 0.08 0.09D-1168 1.98 1.74 D-1170 0.32 0.20 D-1171 1.01 0.56 D-1173 0.64 0.23D-1176 1.48 0.95 D-1206 0.74 0.25 D-1235 0.58 0.35 D-1246 0.17 0.11D-1340 0.31 0.44 D-1364 0.51 0.72 D-1373 0.22 0.32 D-1389 0.19 0.12D-1397 0.47 0.08 D-1408 0.90 0.44 D-1426 0.26 0.43 D-1431 0.41 0.54D-1432 3.39 3.31 D-1438 0.10 0.16 D-1443 0.53 0.28 D-1472 0.57 0.60D-1484 0.29 0.17 D-1494 0.21 0.07 D-1497 1.50 0.15 D-1686 27.78 >83.33D-1708 0.66 0.56 D-1798 0.48 0.52 D-1801 0.50 0.76 D-1811 0.73 0.57D-1813 0.29 0.29 D-1815 0.80 0.24 D-1824 0.39 0.33 D-1826 1.08 0.07D-1832 0.26 0.12 D-1981 1.33 0.46 D-1983 0.05 0.05 D-1989 1.35 0.83D-1999 0.23 0.14 D-2000 0.67 0.45 D-2006 0.52 0.55 D-2045 0.14 0.21D-2056 0.27 0.19 D-2069 1.33 1.33 D-2122 0.80 1.41 D-2142 2.07 1.12D-2143 1.12 0.68 D-2311 0.56 0.46 D-2335 0.28 0.23 D-2357 0.38 0.28D-2361 0.75 0.70 D-2401 0.99 0.76 D-2461 1.05 0.91

All of the Tier 2 siRNA molecules, except for 8 siRNA moleculestargeting the 5′ and 3′ ends of the human ASGR1 transcript, were testedin the in vitro anti-ASGR1 immunoassay in Hep3B cells at two differentconcentrations (1.25 nM and 5 nM). The reduction in ASGR1 cell surfaceexpression relative to expression in cells transfected withnon-targeting siRNAs in Hep3B cells for each siRNA molecule is shown inTable 4 below. Cell count measurements are also provided.

TABLE 4 In vitro efficacy of Tier 2 ASGR1 siRNA molecules in immunoassayscreen Normalized Normalized Alexa 488 Alexa 488 Cell Mean Cell MeanDuplex count Intensity count Intensity No. 5 nM 5 nM 1.25 nM 1.25 nMD-1000 −7.89 −69.66 14.36 −16.59 D-1001 −37.89 −69.18 2.85 −24.50 D-1002−31.79 −57.03 11.54 −1.83 D-1003 −3.36 37.23 9.55 −2.29 D-1004 1.60−59.64 8.80 −8.51 D-1005 3.05 −28.72 −1.24 2.39 D-1006 −32.96 −57.28−7.05 −9.66 D-1007 −27.69 −70.79 14.31 −3.33 D-1008 30.65 9.28 20.7912.22 D-1009 −23.35 −73.69 11.67 −2.29 D-1010 35.43 −55.89 3.56 −22.96D-1012 16.98 −75.78 10.37 −34.40 D-1013 24.10 12.20 8.33 2.11 D-10147.31 −65.21 12.97 −6.17 D-1015 −7.71 −73.25 16.15 −4.04 D-1016 −60.21−61.66 6.81 14.15 D-1017 11.83 −56.24 4.70 2.01 D-1019 29.84 −64.73−1.46 −12.82 D-1020 25.65 −53.00 −2.67 −10.04 D-1021 −26.83 −75.10 2.11−12.78 D-1022 −0.55 −59.91 −3.68 −22.68 D-1023 −0.29 13.64 11.80 2.03D-1025 27.98 −1.15 7.76 −2.93 D-1026 −18.53 −56.89 13.22 8.74 D-102755.59 −36.50 11.10 1.38 D-1028 −32.96 −19.81 5.19 −0.71 D-1029 −20.89−63.26 13.44 −5.46 D-1030 27.16 −43.18 5.36 −9.83 D-1031 6.49 −13.6523.42 2.27 D-1032 −1.16 −10.97 −4.20 −13.77 D-1033 −30.24 −69.98 −0.58−13.92 D-1034 −28.53 −23.08 9.46 3.34 D-1035 25.32 −65.71 3.16 −16.06D-1037 −3.94 −52.11 11.55 −9.25 D-1038 −7.40 −57.60 16.02 −6.46 D-103914.47 −71.31 9.64 −34.96 D-1040 −4.57 −54.21 14.10 −0.10 D-1041 −11.52−69.09 −6.14 −14.98 D-1042 −30.28 −38.43 −4.52 −15.31 D-1043 36.88−29.24 6.55 −7.92 D-1044 2.11 33.99 18.08 14.48 D-1045 −51.56 −57.056.11 −6.29 D-1046 −4.10 −54.90 18.23 4.76 D-1047 2.37 −61.85 10.29 5.28D-1048 47.43 −78.54 22.26 −40.26 D-1049 −21.19 −48.21 18.33 3.60 D-1050−48.89 −66.58 2.19 −5.94 D-1051 −24.11 −78.56 9.89 −3.84 D-1052 20.98−65.51 −1.22 −10.60 D-1053 −24.22 −33.53 16.74 0.96 D-1054 13.12 −66.55−6.19 −20.07 D-1055 −1.47 −30.04 5.24 −6.10 D-1056 −5.32 −74.11 21.67−19.44 D-1057 −4.16 8.07 −2.83 −19.78 D-1058 15.98 −60.00 9.56 −6.36D-1059 48.61 −77.08 1.54 −3.17 D-1060 −15.59 −38.07 4.82 2.38 D-1061−23.58 −28.91 13.89 −4.03 D-1062 6.14 −17.75 38.76 11.62 D-1064 0.34−47.84 4.56 −9.18 D-1065 −6.79 −66.80 6.78 −22.67 D-1066 −65.89 −80.552.45 −7.49 D-1067 −33.03 −50.01 −10.13 −11.57 D-1068 24.92 34.77 12.70−11.13 D-1070 −2.32 −77.56 4.69 −41.44 D-1071 −13.75 −49.58 −4.45 −27.30D-1072 −17.16 −71.02 5.27 −11.02 D-1073 3.53 −70.15 0.08 −20.71 D-1074−2.69 −52.58 −3.32 −13.23 D-1075 −29.75 −69.26 1.62 1.87 D-1076 −19.38−47.86 −12.97 −23.12 D-1077 9.29 −65.14 9.30 −6.34 D-1078 −23.33 −40.075.01 −3.70 D-1079 −42.01 −63.78 0.89 0.06 D-1080 −17.33 −44.28 2.34 2.62D-1081 13.97 −12.08 12.60 0.04 D-1082 31.36 −61.76 −6.47 −45.11 D-1083−4.22 −78.92 24.94 −13.91 D-1084 −0.24 −78.23 0.59 −7.50 D-1085 −11.36−44.47 0.49 −16.32 D-1086 −7.98 −30.62 16.32 1.09 D-1087 −4.69 −48.3114.94 −7.65 D-1088 34.69 −75.14 3.15 −32.99 D-1089 −0.64 14.68 10.290.81 D-1090 24.33 −73.19 1.58 −29.18 D-1092 15.32 −38.05 22.85 7.63D-1093 16.98 −66.40 5.15 −3.92 D-1094 −28.11 −76.79 9.05 −12.11 D-1095−62.01 −80.60 −6.17 −18.12 D-1096 29.08 −59.93 22.01 −14.91 D-1097 −3.49−57.96 13.47 −9.93 D-1098 −4.13 −77.54 18.58 11.13 D-1099 1.50 −37.4313.86 −9.51 D-1100 36.33 −34.40 −4.77 −12.29 D-1101 18.17 −67.20 −3.35−12.05 D-1102 −32.57 −36.16 7.11 −2.90 D-1103 14.19 −66.58 2.19 −4.37D-1104 −26.08 −71.40 −0.66 −21.29 D-1105 26.73 −18.96 3.08 −10.81 D-11064.76 −60.72 7.37 6.57 D-1107 0.97 −31.43 9.38 −3.31 D-1108 −8.06 −74.963.16 −35.88 D-1109 16.36 −42.30 4.53 −4.72 D-1110 12.49 −25.10 24.015.26 D-1111 30.30 −7.13 2.99 −8.86 D-1112 −13.01 −16.74 27.80 11.70D-1113 6.34 −59.24 10.21 −0.62 D-1114 −24.13 −24.51 9.04 −7.34 D-1115−24.09 −75.18 −10.45 −43.54 D-1116 −15.10 −30.34 12.21 −0.66 D-111717.76 9.45 4.90 −8.17 D-1118 2.98 −22.01 21.22 1.82 D-1119 −14.42 −29.69−4.61 −9.83 D-1120 −8.07 −59.22 2.09 −18.67 D-1121 1.37 −38.92 0.00−18.03 D-1122 −25.60 −13.63 −16.07 −5.83 D-1123 −23.76 −13.41 18.49−0.36 D-1124 29.36 −39.05 30.71 8.55 D-1125 15.34 −60.37 −1.83 −18.69D-1126 8.35 −20.20 11.35 14.70 D-1127 13.94 −56.41 4.37 −23.47 D-1128−9.29 −43.79 11.18 7.39 D-1129 −36.45 −70.33 7.05 19.91 D-1130 7.87−58.39 −6.32 −12.47 D-1131 −4.69 −60.73 11.29 −12.42 D-1132 13.53 3.3420.18 10.44 D-1133 6.11 −32.84 10.06 −9.99 D-1134 −8.48 −55.96 5.75−15.23 D-1136 −23.95 −25.01 15.68 −0.43 D-1137 −31.73 −70.89 5.75 −7.02D-1138 25.27 3.96 10.59 −5.05 D-1139 36.30 16.88 9.30 0.56 D-1140 38.80−11.18 8.96 −9.00 D-1141 −9.08 −59.67 1.92 −3.78 D-1142 −2.57 −29.794.60 −9.26 D-1143 10.00 −23.14 18.41 3.98 D-1144 −22.41 −67.81 5.16−4.68 D-1145 −11.01 −39.40 14.98 −5.93 D-1146 20.61 −60.09 5.83 −14.30D-1148 −32.46 −42.55 24.90 13.98 D-1149 −9.39 −20.73 −4.28 −13.98 D-115016.11 −22.00 7.88 −9.56 D-1152 −54.17 −60.99 7.05 −4.58 D-1153 11.52−30.76 −12.21 −20.90 D-1154 −0.73 −46.96 6.97 −6.28 D-1155 7.26 −13.953.23 −13.24 D-1156 3.05 −46.25 13.69 −5.66 D-1157 12.93 −59.47 16.23−11.64 D-1159 −29.47 −55.55 1.70 −4.00 D-1160 −0.42 −73.42 −0.81 −10.76D-1161 18.25 −23.94 6.32 6.22 D-1163 −44.43 −63.99 13.02 −0.59 D-1164−7.43 −49.38 8.03 −6.32 D-1168 1.65 −40.89 4.12 −19.65 D-1169 −19.55−49.51 13.74 −5.68 D-1170 −26.61 −71.74 7.70 −2.54 D-1171 4.11 −75.0122.41 11.35 D-1173 45.33 −49.69 7.69 −11.52 D-1176 −14.04 39.10 14.2310.32 D-1177 −25.04 −68.88 3.57 −5.82 D-1178 18.05 −75.84 9.63 −21.29D-1179 9.45 −28.67 5.52 1.22 D-1180 −11.55 0.73 −1.94 −1.98 D-1181−30.55 −78.57 15.31 −17.01 D-1182 −40.88 −57.42 9.63 −11.33 D-1183 24.63−66.53 5.98 −10.91 D-1184 −13.11 −33.62 8.80 −6.36 D-1185 −16.61 −39.8212.97 5.82 D-1186 19.94 −59.27 9.32 −5.26 D-1187 −0.73 −68.89 −3.98−15.07 D-1188 −32.58 −53.36 −10.13 −28.19 D-1189 −11.28 −72.46 8.12−12.36 D-1190 −13.36 −51.18 6.31 −13.35 D-1191 −18.43 −12.80 8.62 −7.05D-1192 5.78 −70.90 11.63 −38.66 D-1193 −12.63 −39.72 13.11 −2.18 D-1194−7.12 59.76 23.34 19.24 D-1195 −39.18 −71.13 0.73 −0.80 D-1196 19.113.83 20.58 7.12 D-1197 −3.58 −53.14 10.96 −4.07 D-1198 11.37 −60.6244.32 7.61 D-1199 2.66 −40.94 −10.62 −20.63 D-1200 −31.07 −78.56 2.19−15.79 D-1201 −8.03 −18.56 9.05 3.62 D-1202 6.39 19.18 11.88 10.69D-1203 −23.94 −38.26 11.97 −7.76 D-1204 18.53 −34.62 14.06 −0.10 D-120551.91 −39.17 −3.16 −17.83 D-1206 −37.20 −73.16 −3.16 −20.83 D-1207−45.60 −78.35 4.10 −17.84 D-1208 17.42 −61.26 3.15 −27.63 D-1209 0.09−22.29 3.77 1.55 D-1210 −7.52 −0.01 7.11 −3.27 D-1212 −25.06 −80.96−10.57 −28.32 D-1213 −16.69 −70.97 −1.58 −20.96 D-1214 19.94 −18.87−14.23 −21.87 D-1215 −3.19 −64.93 12.37 −10.03 D-1216 −20.89 −56.88 5.19−8.45 D-1217 −4.76 −59.77 −0.32 0.60 D-1218 8.81 −1.56 1.67 −13.50D-1219 8.72 −15.45 5.43 4.40 D-1220 −3.92 −54.00 10.62 −3.14 D-1221−9.45 −78.87 25.02 −19.76 D-1222 24.49 16.25 9.14 −7.22 D-1223 −4.11−38.73 16.18 3.39 D-1224 5.78 −63.52 11.05 −0.39 D-1225 −27.52 −39.1212.80 −6.49 D-1226 25.32 −71.53 3.43 −7.69 D-1227 −2.40 −60.87 22.203.16 D-1228 20.18 −11.50 25.69 15.83 D-1229 −21.59 −40.96 7.68 −3.08D-1230 −6.72 −60.01 8.80 −12.19 D-1231 −11.10 −17.77 11.88 −0.38 D-1232−23.04 −56.57 4.37 −16.21 D-1233 −46.59 −69.92 4.90 −24.43 D-1235 6.78−71.94 8.16 −14.27 D-1236 −8.53 −53.49 23.18 1.28 D-1238 31.17 −33.381.70 −12.57 D-1239 15.59 −59.21 −6.06 −21.61 D-1240 14.47 −42.60 4.23−12.99 D-1241 −3.73 −15.54 6.14 −4.81 D-1242 −27.39 −44.26 11.51 12.06D-1243 26.82 −64.75 7.28 −30.55 D-1244 −36.97 −53.39 −8.20 −18.44 D-124513.97 −59.19 6.09 −5.81 D-1246 12.21 13.16 −1.62 −24.75 D-1247 −12.88−32.06 3.88 −6.32 D-1248 −38.80 −68.71 16.29 12.31 D-1249 −1.75 6.9214.79 −13.93 D-1251 30.27 −79.34 7.61 −20.01 D-1252 2.23 −50.19 4.37−3.52 D-1253 −32.85 −29.67 2.99 2.33 D-1254 −36.45 −76.52 8.02 −21.55D-1255 −3.39 −64.64 −4.77 −14.70 D-1256 1.01 −3.30 25.61 14.58 D-1258−49.01 −54.26 4.65 −6.34 D-1259 −21.83 −49.68 9.72 −2.72 D-1260 23.21−65.48 8.03 −4.15 D-1261 −31.64 −57.04 −9.89 −13.26 D-1262 −29.72 −66.74−5.61 15.96 D-1263 −8.33 −65.33 0.08 −11.83 D-1264 36.01 −20.16 4.61−14.46 D-1265 −1.65 −51.45 −3.48 −6.13 D-1266 −13.01 −19.17 −5.06 −8.27D-1267 −5.05 −31.71 11.97 −0.60 D-1268 1.11 10.61 0.33 −5.81 D-1269−5.56 −53.51 10.62 −3.13 D-1270 1.10 −60.09 17.57 −2.00 D-1271 −5.05−68.78 −0.50 −21.76 D-1272 −14.37 −50.90 −1.83 −18.13 D-1273 −31.36−62.83 15.04 −1.17 D-1274 −7.69 −21.09 9.88 −0.30 D-1275 25.07 −72.645.58 −25.02 D-1276 −4.59 −35.87 −3.35 −11.79 D-1277 46.09 −35.72 −3.63−25.23 D-1278 8.53 −70.41 14.59 −19.50 D-1279 5.96 −4.96 22.09 28.38D-1280 −14.29 −58.65 7.86 −3.32 D-1281 10.07 −32.98 8.49 0.81 D-1282−28.56 −74.04 −3.80 −35.39 D-1283 −18.30 −34.79 9.14 2.84 D-1284 −12.11−36.83 11.05 4.28 D-1287 11.71 −21.34 −12.29 −28.09 D-1290 −2.13 −78.34−3.56 −11.58 D-1292 −23.43 −28.70 −0.57 −16.42 D-1295 25.46 −57.29 15.12−1.18 D-1297 3.21 −63.31 25.77 22.14 D-1298 −3.92 −68.03 −9.71 −34.14D-1300 −10.33 −49.32 0.97 2.06 D-1304 21.30 −10.48 −1.29 −11.82 D-130541.65 13.01 8.80 −1.54 D-1306 29.33 −7.63 8.81 −6.33 D-1307 22.31 −29.34−1.52 −13.70 D-1308 15.60 −20.06 7.53 −4.85 D-1309 −14.33 −52.52 16.98−9.89 D-1310 −6.36 −76.66 2.35 −10.23 D-1311 14.29 −65.01 −12.32 −29.59D-1312 −8.48 −79.39 −0.42 −14.88 D-1313 −44.04 −74.66 0.67 −4.61 D-1314−11.91 −43.05 10.02 −5.56 D-1315 8.16 −46.22 −0.32 2.27 D-1316 17.1727.34 −1.49 −3.64 D-1317 −2.23 −55.09 5.58 −11.09 D-1318 11.27 −47.3218.80 −7.48 D-1319 14.66 −25.03 16.29 13.59 D-1320 19.79 −0.18 8.88−7.73 D-1321 9.10 −76.11 17.02 −0.59 D-1322 −44.56 −69.34 3.57 −13.82D-1323 28.21 −15.42 0.41 −11.61 D-1324 21.72 −62.51 −10.04 −6.03 D-132518.72 0.07 6.16 4.25 D-1326 −4.10 −72.25 −19.37 −33.56 D-1327 16.07−68.74 2.99 −16.27 D-1328 20.37 −69.18 −2.32 −18.28 D-1329 −25.32 −44.99−3.16 3.98 D-1330 −16.33 −63.82 −5.69 −16.83 D-1331 0.99 −35.15 11.59−7.43 D-1332 −15.23 −53.20 23.43 7.37 D-1333 −11.93 −39.12 −2.27 −4.03D-1334 −47.27 −65.62 9.21 −3.59 D-1336 3.44 −18.99 12.97 3.86 D-13374.67 −67.99 4.05 −14.59 D-1338 10.50 −20.32 −13.20 −27.03 D-1339 10.45−57.10 18.11 −5.50 D-1340 7.50 −75.18 −11.35 −51.99 D-1341 −14.13 −42.797.28 −2.05 D-1343 32.45 −74.74 25.44 −8.83 D-1344 21.49 −79.31 8.57−41.53 D-1345 5.56 −71.98 4.40 −14.59 D-1346 10.84 −47.45 −2.43 −21.79D-1347 11.85 −38.12 6.72 −16.62 D-1348 −4.77 −76.11 −1.17 −20.86 D-1349−36.51 −61.19 10.46 11.03 D-1351 −15.32 −73.27 4.78 −8.76 D-1352 10.36−63.16 3.64 −5.33 D-1353 1.21 −8.18 5.23 −10.04 D-1354 3.34 −75.94 4.23−15.74 D-1355 7.71 11.27 19.92 11.87 D-1356 −10.85 −63.81 6.34 0.13D-1357 1.31 −37.05 −14.02 −16.27 D-1358 24.49 41.10 28.13 20.76 D-13595.56 −23.75 13.20 −2.77 D-1360 17.08 37.58 1.08 −11.99 D-1361 −22.77−47.82 7.21 −9.44 D-1362 19.55 −59.93 8.97 −23.28 D-1363 −51.44 −77.047.78 −22.46 D-1364 23.06 −10.37 9.40 −4.87 D-1366 −9.36 −51.45 5.94 6.52D-1368 −2.29 −52.35 11.55 −12.22 D-1369 −9.85 −46.01 −7.70 −10.96 D-1370−41.93 −78.44 12.97 −28.38 D-1371 −17.16 −36.27 4.02 −4.25 D-1372 −19.19−63.22 10.94 7.71 D-1373 22.27 −76.68 0.97 −24.18 D-1374 −24.95 −73.678.54 −38.43 D-1375 −38.52 −63.20 2.67 −9.04 D-1377 −59.82 −77.46 9.62−16.34 D-1378 17.43 −27.46 0.08 −1.36 D-1379 39.59 −77.99 8.65 −21.47D-1380 16.50 −68.50 −2.57 −24.69 D-1381 5.13 −34.48 0.49 −0.75 D-1382−21.97 −80.36 12.13 −23.38 D-1383 −4.29 −46.09 16.86 1.03 D-1384 1.74−78.01 14.64 −24.12 D-1385 −26.66 −15.55 9.21 4.44 D-1386 −2.76 −22.47−9.79 −16.76 D-1388 11.93 −28.38 16.90 18.95 D-1389 −35.23 −75.62 13.473.46 D-1391 −9.54 −55.57 1.76 8.01 D-1392 −25.60 −69.12 −6.44 −31.51D-1393 4.95 −72.52 4.21 0.75 D-1394 7.16 −77.18 8.33 −7.38 D-1395 30.60−72.67 3.81 −29.55 D-1396 −7.43 −77.70 2.43 −28.95 D-1397 25.07 −80.005.34 −32.68 D-1401 0.05 6.64 −11.54 −12.09 D-1402 −14.85 −66.16 0.32−2.45 D-1404 1.69 −41.85 17.51 0.44 D-1408 32.66 −74.78 5.23 −9.30D-1409 −10.30 −74.45 −1.58 −32.92 D-1410 −37.77 −55.14 9.16 4.08 D-1411−9.71 −61.25 5.83 −11.94 D-1412 −4.40 −62.34 15.06 1.14 D-1413 −38.99−26.13 8.91 13.59 D-1414 0.68 −48.38 11.08 −5.56 D-1415 30.11 −79.9414.79 −8.31 D-1416 9.00 −72.55 −8.25 −27.91 D-1417 20.52 −1.74 14.234.17 D-1418 −23.30 −66.73 15.23 −0.48 D-1419 9.36 −78.21 13.31 −33.52D-1420 −25.30 −63.31 −10.54 −25.54 D-1421 −36.82 −71.74 −7.14 −31.17D-1422 −1.31 −72.73 0.50 −31.24 D-1423 6.97 −32.14 5.77 −0.11 D-1424−0.05 −25.55 3.32 −13.94 D-1425 14.42 −32.00 −3.96 −15.93 D-1426 −10.30−22.92 13.20 2.02 D-1427 −8.53 −61.49 11.51 −10.59 D-1428 19.28 −18.075.43 −4.47 D-1429 5.81 −78.39 3.48 −41.33 D-1430 9.87 −77.50 −2.26−28.08 D-1431 −20.64 −79.16 2.76 −35.67 D-1432 −12.59 −48.84 7.21 −14.06D-1433 −18.88 −77.44 −0.49 −19.75 D-1434 30.88 −31.72 25.22 7.46 D-14353.97 −79.61 2.51 −31.91 D-1436 8.08 −71.76 −1.08 −28.30 D-1437 −17.56−72.82 15.27 8.76 D-1438 −13.50 −49.08 1.49 −14.23 D-1439 21.43 −63.7017.18 −0.95 D-1440 −0.52 −65.90 5.51 −21.92 D-1441 0.09 −38.28 3.85−9.17 D-1442 2.66 −55.33 −3.43 −26.16 D-1443 −24.24 −30.45 11.54 −0.89D-1444 33.58 −65.83 24.85 −0.39 D-1445 −22.79 −16.47 14.20 −7.26 D-144622.30 −16.81 8.46 −8.89 D-1447 6.18 −68.19 16.29 −11.29 D-1448 4.01−49.66 1.54 −7.00 D-1449 −40.41 −15.22 3.08 −10.87 D-1450 −0.77 −77.989.78 −23.68 D-1451 −6.58 −69.35 20.12 −8.13 D-1456 −16.75 −79.94 2.10−14.80 D-1457 −17.33 −80.91 3.56 −39.89 D-1458 26.04 −29.22 8.57 −4.40D-1459 −12.84 −76.97 9.47 −15.64 D-1460 −3.29 −76.01 −4.45 −25.68 D-1461−5.61 −77.92 −1.70 −19.63 D-1462 2.08 −63.16 −6.72 −26.68 D-1463 −16.61−67.89 1.09 2.56 D-1464 −8.48 −54.92 8.11 −14.13 D-1465 5.76 −71.0918.59 0.96 D-1466 20.46 −37.62 23.18 −2.68 D-1467 20.32 −13.04 −17.34−15.71 D-1468 31.45 −64.41 −2.19 −4.79 D-1469 12.68 5.48 −7.28 −20.26D-1470 6.92 −66.88 9.38 −12.70 D-1471 −22.22 −74.37 13.02 −13.21 D-1472−11.81 −79.47 15.36 −19.74 D-1473 −38.46 −72.40 −0.17 −21.43 D-1474 2.97−57.92 −5.19 −16.96 D-1475 −16.93 −44.54 3.65 2.15 D-1476 −5.89 −52.6712.80 −3.85 D-1477 −17.89 −60.62 −3.68 −2.20 D-1480 28.79 10.79 −3.24−16.12 D-1481 6.70 −41.49 3.35 −21.34 D-1482 7.43 −19.68 17.91 6.02D-1483 1.98 −21.91 4.90 −5.20 D-1484 −16.51 −55.79 1.51 6.85 D-1485−36.43 −80.11 9.81 −11.66 D-1486 5.42 −62.97 5.50 −13.09 D-1487 10.89−23.86 −5.81 −11.30 D-1488 −14.71 −68.13 6.06 −5.50 D-1489 −21.55 −76.48−5.83 −18.03 D-1490 26.04 −80.46 0.57 −46.23 D-1491 32.72 −80.10 1.78−26.10 D-1492 39.08 −37.64 6.69 4.14 D-1493 3.54 −74.86 14.02 −7.60D-1494 −19.75 −43.37 20.10 2.38 D-1496 −16.08 −70.55 4.54 −21.74 D-149732.26 −81.52 17.58 −27.07 D-1498 12.11 −40.01 51.46 25.60 D-1499 −24.39−79.00 2.34 4.18 D-1500 −51.56 −70.59 5.02 −16.85 D-1501 46.47 −65.12−19.40 −28.47 D-1503 −31.92 −69.56 9.89 −10.48 D-1504 5.50 −37.04 22.855.61 D-1507 8.48 −55.73 23.50 −8.71 D-1508 −11.65 −68.55 12.47 −16.00D-1509 −17.76 −55.16 2.07 −15.48 D-1510 −4.36 −29.45 7.68 −3.23 D-15120.33 −33.83 6.40 5.03 D-1513 −40.09 −64.18 17.24 −2.88 D-1514 −19.65−33.00 1.86 −10.55 D-1515 −24.63 −55.52 3.73 0.05 D-1517 −47.61 −74.578.95 −9.97 D-1519 11.56 −29.26 −12.37 −18.89 D-1520 32.24 3.14 15.60−0.57 D-1522 32.14 8.88 0.97 −13.70 D-1523 −7.43 −50.43 −1.00 −11.45D-1524 15.49 −8.69 2.02 0.13 D-1525 −22.79 −63.32 13.36 7.28 D-152610.45 −12.02 −9.30 −17.56 D-1527 −7.16 −39.49 14.64 10.43 D-1528 30.7816.18 −8.65 −26.51 D-1529 5.41 −34.44 5.52 9.61 D-1530 23.04 −14.77 2.10−12.78 D-1531 36.53 −1.44 0.50 −11.53 D-1532 −11.19 −22.94 18.16 2.41D-1533 11.38 −70.75 7.78 −6.32 D-1541 −3.76 −50.19 30.21 23.94 D-15424.74 −17.47 9.54 1.27 D-1543 11.93 −76.40 7.70 −42.72 D-1544 −42.33−52.93 14.69 −0.38 D-1545 −9.67 −41.93 4.13 −9.45 D-1546 −14.76 −71.939.54 −7.00 D-1547 −45.14 −63.59 10.29 −7.72 D-1548 7.53 −32.51 −6.85−8.07 D-1549 12.78 −59.45 15.20 −24.86 D-1550 5.96 −73.04 23.10 −3.15D-1551 −3.17 −50.57 −4.48 −10.38 D-1552 17.08 −3.57 3.49 −13.28 D-1553−37.67 −70.41 −11.02 −23.50 D-1555 −19.85 −47.96 1.54 −8.57 D-1557−22.96 −65.81 4.46 −6.26 D-1558 15.05 −10.38 16.99 13.12 D-1559 −11.13−11.60 10.59 −5.72 D-1560 −14.95 −70.17 10.29 −4.46 D-1561 −2.81 −53.50−3.31 −23.23 D-1562 −0.90 −70.55 8.91 −2.19 D-1563 6.74 −36.12 −1.70−11.00 D-1564 −0.33 −76.38 9.89 −9.36 D-1565 11.74 −70.09 17.57 3.84D-1566 −19.26 −36.08 4.12 −20.01 D-1567 −36.21 −26.16 16.33 8.05 D-1568−22.17 −29.62 8.89 −0.71 D-1569 21.63 −2.09 10.46 −5.42 D-1570 22.11−76.43 14.14 −32.88 D-1571 −0.82 −37.92 5.89 −14.85 D-1573 −10.18 −64.734.44 −1.95 D-1574 −2.61 −35.69 2.26 −15.60 D-1575 15.97 −5.44 −9.22−15.32 D-1576 −10.11 −49.40 −16.68 −16.01 D-1577 −36.79 −70.90 22.183.36 D-1579 −13.84 −42.68 7.44 −10.53 D-1580 −16.15 −0.78 11.21 1.18D-1582 −29.33 −70.31 4.61 −9.18 D-1583 −3.91 −72.50 −5.83 −22.00 D-1584−17.49 −69.74 14.91 −4.97 D-1585 29.70 −51.61 6.85 −9.20 D-1586 −10.36−24.80 −3.64 −14.72 D-1587 −12.24 −16.84 −4.32 −14.95 D-1588 0.39 −1.9513.34 −2.91 D-1589 40.88 0.52 14.26 6.47 D-1590 −5.61 22.64 −0.97 −7.11D-1591 2.31 −30.67 −3.48 −13.93 D-1592 −19.75 −38.53 3.55 −6.58 D-159319.01 6.07 19.17 16.66 D-1594 −17.20 −57.14 −1.44 −15.06 D-1595 −15.97−57.16 19.00 7.35 D-1596 −15.98 −42.84 9.16 8.17 D-1597 1.66 −71.11 7.27−23.62 D-1598 −4.22 −63.19 7.03 −6.96 D-1599 11.04 −39.72 −1.13 −20.46D-1600 17.62 −38.11 9.86 −16.81 D-1601 35.77 −77.17 11.75 −26.16 D-1602−33.82 −69.95 5.89 −13.32 D-1603 31.45 −71.55 −2.35 −30.03 D-1604 20.89−1.24 −1.30 −9.64 D-1605 6.55 −53.67 3.48 −9.38 D-1606 2.69 −23.77 2.27−1.67 D-1607 −17.91 −51.36 6.31 10.90 D-1608 27.20 −32.06 18.56 3.72D-1609 18.06 −19.53 7.54 −5.72 D-1610 0.29 −5.19 −1.21 −14.22 D-16117.55 36.89 5.34 −0.83 D-1612 −12.84 −19.84 24.77 13.24 D-1613 −12.97−75.43 19.45 −2.76 D-1614 41.14 37.89 14.31 −4.57 D-1615 27.30 −41.7423.10 14.97 D-1616 12.94 3.94 24.94 30.37 D-1617 −12.74 −28.74 11.92−7.36 D-1618 −21.59 −33.88 21.75 9.98 D-1620 12.43 −59.95 −6.31 −6.82D-1621 −26.08 −48.87 −11.78 −11.89 D-1622 −11.28 6.47 13.22 4.49 D-162411.89 −55.62 15.64 −2.82 D-1625 −23.14 −30.97 13.42 −0.85 D-1626 −48.99−63.51 17.15 −3.64 D-1628 −1.56 −38.82 −1.09 −13.60 D-1629 22.68 −43.27−2.76 −9.65 D-1630 42.94 −59.46 13.31 −0.07 D-1631 −7.87 −61.54 −5.43−11.47 D-1632 31.45 40.65 3.40 1.39 D-1633 −1.02 −42.12 2.32 −10.80D-1634 9.14 −31.08 16.35 −8.94 D-1635 −21.74 −62.17 3.57 −6.98 D-163619.09 −24.92 8.88 −24.34 D-1637 24.00 −50.62 −9.16 −13.04 D-1638 −26.95−66.99 10.06 −9.12 D-1639 1.01 −36.90 8.95 −4.91 D-1640 −11.91 −64.78−0.16 −9.43 D-1641 4.60 −48.90 5.64 −16.05 D-1642 0.58 −41.02 18.35 1.62D-1643 −8.85 −76.54 9.54 −15.54 D-1644 10.51 −52.89 20.58 14.19 D-164512.87 −71.23 12.16 −0.54 D-1646 −5.78 −74.08 −4.69 −27.01 D-1647 −4.45−5.92 6.63 −8.13 D-1648 13.46 34.26 22.88 4.87 D-1649 −47.63 −56.76 4.128.02 D-1650 32.58 −59.12 4.05 −9.81 D-1651 1.84 −64.00 −3.73 −17.15D-1652 15.30 −41.66 4.28 −10.21 D-1654 −5.23 −33.75 3.00 3.66 D-16556.87 −42.73 14.47 8.55 D-1657 −2.37 −52.43 4.98 −15.30 D-1658 2.03−18.81 19.00 8.79 D-1659 −9.57 −59.55 23.74 −15.69 D-1660 −26.48 −74.5519.78 −2.82 D-1661 17.81 −38.33 41.55 0.20 D-1662 14.45 −52.42 −0.59−27.08 D-1663 4.07 −58.51 2.59 −16.05 D-1664 15.20 −53.44 6.39 −8.56D-1665 −23.06 −68.84 21.96 14.62 D-1666 13.84 −40.77 3.64 2.18 D-1667−24.50 −45.47 −9.04 6.51 D-1668 6.61 −78.38 9.62 −24.13 D-1669 25.32−65.31 27.36 −27.95 D-1670 9.53 20.77 −11.62 −24.98 D-1671 14.18 −38.83−1.66 −12.03 D-1672 10.69 −42.66 −1.83 −19.00 D-1673 −6.87 −12.52 12.45−2.14 D-1674 −3.44 −30.88 17.10 12.97 D-1675 −41.19 −75.58 12.13 −15.82D-1676 −12.87 −60.60 15.24 −7.71 D-1677 −13.84 −38.01 2.26 −9.77 D-16796.72 −42.72 10.87 1.76 D-1680 −0.82 −58.97 2.49 −4.77 D-1681 2.40 −70.083.89 −16.35 D-1682 −22.79 −48.92 10.87 −4.47 D-1684 16.50 −50.83 −2.16−13.61 D-1685 −16.42 −79.02 7.62 −61.49 D-1686 4.79 −74.35 6.72 −13.30D-1688 −16.27 −49.27 −6.81 −19.80 D-1689 3.43 −73.67 0.91 −18.82 D-1690−22.68 −21.68 −5.75 −7.62 D-1691 15.82 −72.63 0.75 −33.93 D-1692 −8.53−65.87 23.18 −2.19 D-1693 −6.53 −52.63 2.49 −13.30 D-1695 18.62 −73.010.73 −16.64 D-1696 −27.53 −41.20 12.86 11.56 D-1697 −0.63 −76.21 33.11−5.40 D-1698 10.73 5.15 24.35 24.10 D-1699 7.59 −49.15 −2.19 −15.43D-1700 −5.47 −42.44 24.90 13.63 D-1701 7.02 −9.16 9.08 5.69 D-1702−47.80 −24.11 13.81 −4.71 D-1703 −2.85 −61.94 9.13 −4.88 D-1704 −27.90−8.32 9.89 −13.37 D-1705 −37.01 −46.48 3.32 −9.17 D-1706 −19.28 −32.881.70 −8.36 D-1707 7.69 −21.74 9.05 −6.05 D-1708 −16.50 −57.74 6.64 −3.44D-1710 −16.79 −65.45 6.80 −10.38 D-1711 −28.36 −56.25 7.76 −4.87 D-171217.37 −18.44 3.32 −13.05 D-1714 26.79 −33.16 7.78 −4.00 D-1715 4.22−53.78 11.83 −10.35 D-1717 0.87 −57.74 7.92 −3.01 D-1718 35.62 −38.40−0.40 −11.07 D-1720 −5.71 −66.00 5.50 −9.46 D-1721 29.47 45.96 20.4215.01 D-1723 9.72 −28.91 0.00 −30.34 D-1724 −9.58 −71.18 3.80 −9.56D-1725 −12.29 −58.30 5.17 −5.90 D-1726 18.53 8.84 8.13 −5.66 D-1727−8.35 −79.86 14.31 −27.31 D-1728 −14.50 −78.33 6.36 −27.01 D-1729 17.39−6.03 29.92 11.54 D-1730 −45.31 −77.60 20.83 1.51 D-1731 15.79 −18.181.78 −9.77 D-1732 −16.21 −4.72 17.10 4.67 D-1733 −0.64 −75.43 17.74−13.23 D-1735 16.55 −62.74 7.52 −11.28 D-1736 −19.47 −52.32 −3.00 −9.23D-1737 5.41 −9.57 25.94 15.76 D-1738 5.35 −35.18 15.13 −10.09 D-1739−10.18 −57.04 15.48 −15.92 D-1740 10.75 −73.85 0.42 −21.93 D-1742 13.07−19.18 −4.93 −6.50 D-1743 −2.56 −27.26 −4.73 −11.73 D-1744 −7.60 −70.9410.71 −1.73 D-1745 −1.21 −44.21 1.16 −15.57 D-1746 −19.08 −74.88 14.64−18.38 D-1747 6.70 16.51 0.42 −12.56 D-1749 2.52 −40.74 10.35 2.28D-1751 2.95 −76.84 7.05 −18.50 D-1753 2.02 15.57 12.97 19.69 D-175417.30 −1.58 −7.05 −21.69 D-1755 −20.55 −74.51 8.79 −16.00 D-1756 −45.60−12.19 16.17 −1.67 D-1757 −26.04 −61.16 12.21 −7.81 D-1758 −0.34 −73.9747.63 −8.80 D-1759 −63.51 −69.49 −24.07 −41.37 D-1760 5.76 −43.12 −0.91−9.97 D-1761 42.43 −57.23 18.09 8.26 D-1762 0.10 −69.14 10.67 −6.73D-1763 −5.81 −62.90 34.11 14.42 D-1764 6.21 −72.39 5.92 −22.06 D-1765−11.10 −37.69 −1.76 −2.05 D-1766 0.52 4.89 14.20 −11.31 D-1767 18.43−46.54 19.50 0.02 D-1768 12.02 −74.19 2.92 −14.64 D-1769 −0.34 −55.25−2.90 −21.94 D-1770 3.43 −35.46 1.33 −9.24 D-1771 −1.18 −35.96 18.93−2.23 D-1772 −32.57 −78.62 60.42 3.61 D-1774 24.50 −76.51 20.00 −23.79D-1775 21.92 −1.67 20.58 3.16 D-1776 −0.64 −69.08 3.51 −14.00 D-177814.85 −43.29 7.13 3.09 D-1779 45.23 9.53 7.47 −2.55 D-1780 6.36 −53.1216.21 5.81 D-1781 −9.63 −55.20 3.01 −13.99 D-1782 −7.40 −37.94 −2.35−13.41 D-1783 −11.80 −39.91 15.38 −0.50 D-1784 −27.14 −62.76 7.72 −15.32D-1785 −24.69 −56.81 0.57 −20.48 D-1786 −53.14 −57.69 7.62 −13.19 D-1787−1.98 −67.56 14.61 −14.64 D-1788 22.85 −27.66 11.40 −4.65 D-1789 −57.57−60.25 3.65 −2.84 D-1790 18.68 −77.95 16.25 −18.72 D-1791 −7.31 −74.7615.24 −6.11 D-1793 28.65 16.33 12.45 5.71 D-1794 −17.68 −20.87 1.46−7.31 D-1795 17.95 −73.95 −1.74 −27.64 D-1796 5.95 −23.80 7.30 −7.46D-1797 −6.72 −51.09 11.70 −3.05 D-1798 12.40 −2.59 −8.59 −8.33 D-17998.91 −59.33 −2.27 −19.09 D-1800 18.52 17.09 2.45 −4.63 D-1801 −18.92−22.39 11.12 −12.94 D-1802 −3.58 −78.22 2.34 −13.75 D-1803 7.69 −66.70−0.66 −17.72 D-1804 −22.94 −78.51 16.40 −15.98 D-1805 12.14 −36.01 17.769.49 D-1806 −10.65 −73.01 −1.86 −36.31 D-1809 −2.23 −62.22 −3.38 −12.14D-1810 −2.27 −50.18 −7.88 −12.36 D-1811 20.62 −78.33 10.83 −4.71 D-18126.46 −63.75 15.80 5.89 D-1813 26.26 −34.46 3.24 −5.20 D-1815 21.72 24.33−0.91 −15.26 D-1816 −1.27 −62.68 0.65 −15.01 D-1817 −37.56 −48.82 −8.49−10.71 D-1818 −20.79 −49.64 0.32 −14.74 D-1820 1.94 −66.41 −12.77 −12.94D-1821 −27.34 −55.96 −5.69 −15.96 D-1822 15.72 −13.71 5.89 −8.21 D-182336.98 −54.48 7.03 −7.59 D-1824 15.05 −38.99 12.20 −4.28 D-1825 −17.23−62.99 17.95 −0.48 D-1826 −1.74 −79.64 8.73 −30.66 D-1827 −9.29 −36.070.00 −8.87 D-1828 32.08 −63.24 −1.16 −20.15 D-1829 −12.97 −80.27 17.54−16.16 D-1830 −18.62 −21.58 0.81 −16.10 D-1831 −25.78 −74.69 16.99−12.92 D-1832 9.24 16.76 −10.29 −21.96 D-1833 31.56 −68.81 13.34 −1.42D-1834 −10.26 −74.52 −1.21 −27.17 D-1835 −12.48 −67.28 3.77 0.84 D-1836−13.17 −7.34 20.13 17.39 D-1837 −11.36 −20.19 3.48 4.61 D-1838 −14.31−32.03 14.06 1.71 D-1839 10.51 −63.37 11.43 −5.04 D-1841 −16.26 −72.93−5.25 −20.49 D-1843 −46.20 −72.45 7.14 −13.01 D-1844 −4.79 −42.61 15.609.77 D-1845 35.78 −30.99 15.31 −11.75 D-1846 30.49 −44.75 4.53 −10.21D-1847 13.03 −44.91 9.72 −12.37 D-1849 4.40 −50.27 4.32 −11.51 D-185022.87 −19.62 9.48 2.97 D-1851 10.16 −50.32 10.83 −2.40 D-1852 −2.03 3.306.39 −10.53 D-1853 8.76 −34.41 11.54 −0.96 D-1854 −10.75 −32.54 1.86−1.55 D-1855 6.49 −75.79 9.86 −13.88 D-1856 −11.66 −70.52 −4.07 −27.14D-1857 −7.50 −2.38 13.44 −5.69 D-1858 13.63 −54.80 2.27 −14.00 D-1859−17.17 −75.64 6.56 −17.77 D-1860 6.58 −76.19 −5.16 −13.59 D-1861 19.57−39.30 16.86 13.06 D-1862 −5.71 −73.94 4.85 −8.72 D-1863 −47.23 −81.6641.00 −8.60 D-1864 23.90 −75.44 −0.16 −26.92 D-1865 −2.85 −33.66 17.34−7.39 D-1866 −41.34 −73.70 4.20 −7.31 D-1867 7.01 −43.86 23.49 −4.81D-1868 27.33 −45.04 −15.44 −23.60 D-1869 −2.02 −38.29 16.32 0.19 D-1870−3.25 −72.86 −3.89 −33.61 D-1871 −6.24 −59.17 6.53 −8.23 D-1872 6.0612.20 27.36 18.31 D-1873 −19.65 −62.34 3.15 −7.44 D-1875 11.66 −45.276.22 −6.88 D-1877 28.40 −51.53 4.40 −19.08 D-1878 −13.50 −29.09 5.98−8.53 D-1879 1.09 −29.39 12.09 −9.43 D-1880 −2.56 −47.80 −0.17 −6.36D-1881 58.17 −50.55 −2.26 −19.24 D-1882 −26.82 −47.71 2.51 10.76 D-1883−22.50 −13.68 −13.61 −21.06 D-1884 −2.70 −35.00 19.02 −4.66 D-1885 −7.50−73.19 −4.56 −34.52 D-1886 −8.82 −20.72 6.89 11.71 D-1887 11.23 −66.611.94 −17.52 D-1888 −4.79 −17.84 6.06 −4.70 D-1889 7.59 −57.31 15.07−11.89 D-1890 −32.37 −68.00 −4.48 −15.63 D-1891 38.80 −18.11 5.75 −12.54D-1892 3.63 −9.46 9.72 12.53 D-1893 5.33 −62.04 13.70 8.34 D-1894 −16.08−66.91 −3.48 −18.47 D-1895 −39.37 −54.73 10.65 −3.04 D-1896 −34.17−68.67 20.94 2.68 D-1897 −12.55 −79.14 −1.18 −30.09 D-1898 −6.96 −68.28−1.78 −18.90 D-1899 20.72 26.23 −11.80 −14.61 D-1900 −3.73 −64.22 −2.82−23.57 D-1901 6.36 −58.44 5.35 −2.99 D-1902 1.26 21.40 23.69 10.04D-1903 3.94 −74.24 −0.25 −14.16 D-1904 0.68 50.23 2.51 −9.54 D-1905−19.08 −76.82 10.88 −17.12 D-1906 17.08 −43.53 9.46 −4.79 D-1907 −19.00−52.03 0.34 −20.98 D-1908 −21.93 −49.22 18.58 14.88 D-1909 17.59 12.470.41 −5.27 D-1910 12.10 −37.19 2.43 −10.38 D-1911 4.31 −60.58 19.92 7.26D-1912 −7.69 −34.13 2.59 −5.43 D-1913 4.68 −7.14 21.84 10.42 D-1914−9.95 −40.94 20.02 0.05 D-1915 −9.20 −12.41 0.40 −10.13 D-1916 −90.09−69.70 −1.42 10.86 D-1917 7.80 −21.88 21.09 3.31 D-1918 −13.11 −77.486.72 −61.74 D-1919 0.14 −38.79 −5.67 −17.31 D-1920 −29.72 −58.58 19.839.16 D-1921 −12.68 −59.75 1.05 −14.09 D-1922 −19.56 −54.13 6.68 −10.08D-1923 −5.61 −43.21 16.29 12.59 D-1924 12.82 −72.06 17.18 −2.29 D-192516.97 −17.78 19.41 −2.83 D-1926 −15.49 −11.77 68.39 −20.57 D-1927 11.324.87 −4.40 −23.95 D-1928 −6.06 −28.46 24.85 9.59 D-1930 −6.58 −79.2217.16 −29.12 D-1931 −3.39 −62.32 5.61 −16.80 D-1932 9.76 −2.23 10.057.37 D-1933 4.31 −53.82 5.06 −1.69 D-1934 −14.29 −22.36 15.56 8.75D-1935 −3.85 −49.95 2.34 −6.40 D-1936 −9.91 22.02 0.59 −6.11 D-1937−3.73 −61.11 4.32 −21.53 D-1938 −35.33 −77.39 −7.36 −22.69 D-1939 11.467.47 3.24 −6.37 D-1940 −42.29 −45.97 12.47 −5.61 D-1941 −8.16 −45.74−2.27 −7.42 D-1942 −3.00 −55.85 16.65 −10.14 D-1943 33.68 −31.48 3.72−12.52 D-1944 11.42 −58.08 3.04 −16.01 D-1945 −31.07 −68.98 10.70 −6.84D-1946 −36.79 −48.50 19.41 12.95 D-1947 5.52 −18.81 −7.54 −11.92 D-19480.24 −72.84 12.53 −1.26 D-1949 22.59 8.84 14.94 −0.84 D-1950 19.69 13.830.08 −9.03 D-1951 −23.67 −77.04 2.43 −12.37 D-1952 −25.79 −72.15 7.294.56 D-1953 −41.63 −54.76 −4.37 −19.62 D-1954 −16.50 −74.90 26.47 −9.31D-1955 −21.01 −16.60 16.01 9.73 D-1956 −8.95 −74.13 13.94 −22.70 D-1957−15.05 −73.45 19.25 −5.72 D-1958 −17.04 −42.99 −8.57 −21.64 D-1959 −0.24−74.53 10.37 −52.59 D-1960 5.66 −39.86 0.50 −6.53 D-1961 −15.14 −39.354.27 3.43 D-1962 15.69 −64.81 3.10 −12.12 D-1963 0.62 −55.52 −5.16−14.03 D-1964 −16.11 −8.15 4.15 0.01 D-1965 18.91 −68.13 16.29 5.58D-1966 7.31 −66.61 18.67 2.02 D-1967 −31.59 −27.69 −1.33 −18.19 D-196814.95 −50.53 10.04 2.00 D-1969 −39.37 −49.73 11.83 3.08 D-1970 −30.04−71.01 10.46 −3.76 D-1971 −1.36 −0.35 7.76 −3.39 D-1972 −10.99 92.175.92 8.11 D-1973 −26.54 −38.10 1.22 −7.75 D-1974 14.04 −47.50 8.73 −4.02D-1976 −14.47 −28.64 10.05 2.14 D-1977 13.03 −14.91 6.78 8.02 D-1978−17.61 −72.14 −0.25 −34.74 D-1979 12.20 −66.67 0.40 −20.96 D-1980 4.78−7.88 8.03 −4.08 D-1981 22.46 −78.74 11.96 −23.68 D-1982 7.06 −42.7113.64 −14.16 D-1983 147.43 −45.73 7.46 −4.83 D-1984 −9.43 −56.18 18.858.40 D-1985 −7.26 −78.52 −2.18 −23.49 D-1986 11.55 −54.27 10.78 −0.74D-1988 18.49 −80.12 −1.70 −32.09 D-1989 32.04 −77.89 18.27 2.40 D-199017.46 −18.79 3.90 −13.24 D-1991 −7.01 −59.92 1.99 −11.66 D-1992 −82.84−61.20 −12.64 2.49 D-1993 −9.27 −46.43 −3.01 −10.66 D-1995 −0.19 −75.26−6.87 −30.20 D-1996 −11.66 −50.74 −13.03 −29.47 D-1997 −25.96 −44.39−5.94 −21.93 D-1998 19.63 −42.79 14.23 −2.70 D-1999 3.87 −79.84 21.26−32.79 D-2000 5.27 −19.03 1.00 −3.02 D-2001 −15.70 −39.55 −3.40 −20.23D-2002 −0.19 −12.14 1.13 −15.73 D-2003 −9.36 −42.27 3.51 −10.63 D-200419.17 −13.09 −0.59 −8.71 D-2005 27.82 −62.21 4.40 −23.84 D-2006 7.31−26.36 9.24 3.49 D-2007 −52.29 −25.86 −13.89 0.39 D-2009 15.41 −56.6030.54 4.40 D-2011 5.07 −7.16 0.42 −6.12 D-2012 3.49 14.31 9.62 5.80D-2013 29.17 −28.96 10.62 −13.66 D-2014 −9.43 −56.66 23.92 3.59 D-2015−5.66 −13.26 −4.48 −19.19 D-2016 −7.79 −48.27 6.22 −11.50 D-2017 −29.08−10.09 8.46 6.59 D-2018 −12.29 −42.04 5.01 −18.67 D-2019 −9.38 −27.602.27 3.19 D-2020 5.41 −67.72 14.31 −3.73 D-2022 6.39 −35.46 6.31 −0.92D-2023 19.27 −72.15 26.28 −23.46 D-2024 4.76 −55.12 10.21 8.48 D-202524.01 0.84 5.82 −4.60 D-2026 −9.82 −31.92 13.44 2.43 D-2027 −10.47−39.71 −13.78 −16.75 D-2028 25.85 −25.14 7.11 −6.77 D-2029 9.58 −27.9018.84 −6.92 D-2030 25.17 −4.33 −1.05 −8.23 D-2031 −46.06 −61.25 15.98−1.88 D-2032 −1.28 −54.49 −8.62 −19.41 D-2033 37.48 −27.95 −7.46 −21.48D-2034 20.28 33.72 6.95 3.41 D-2035 −27.81 −53.97 19.10 −7.38 D-2036−9.78 −59.60 8.25 −7.34 D-2037 −16.17 −59.12 14.10 4.42 D-2038 −2.48−78.15 26.11 −3.69 D-2039 −17.52 −77.24 4.94 −0.89 D-2040 −7.25 −54.1111.63 0.72 D-2041 9.57 −11.47 −7.05 −6.10 D-2042 −18.34 −29.19 −1.05−8.78 D-2043 −3.12 −66.07 11.63 1.50 D-2044 −37.56 −32.28 21.91 8.04D-2045 −22.65 −75.89 2.02 −21.05 D-2046 −13.91 −74.75 −1.46 −20.68D-2047 −16.88 −53.39 −9.96 −30.30 D-2048 −13.58 −76.86 16.90 −27.20D-2049 −36.21 −9.07 7.60 5.07 D-2051 2.56 −51.79 3.24 −1.81 D-2052−18.34 −46.56 12.48 15.60 D-2053 20.23 −39.84 3.72 −13.74 D-2055 7.15−78.38 7.69 −15.41 D-2056 −2.39 −26.69 17.99 7.25 D-2057 −3.21 −61.151.76 −13.42 D-2058 15.87 −21.62 3.63 −12.54 D-2060 −48.44 −77.63 6.86−42.35 D-2061 14.50 −79.48 19.67 −3.72 D-2062 −16.59 −70.67 −3.65 −21.31D-2063 −7.01 −76.15 9.71 −28.36 D-2065 −4.40 14.16 14.19 0.97 D-206721.53 3.99 −8.46 −21.68 D-2068 23.43 −75.39 −3.40 −26.63 D-2069 2.42−79.04 22.39 −11.16 D-2070 9.38 −61.91 19.53 −0.10 D-2071 0.52 −65.619.08 7.54 D-2072 −5.23 −78.42 7.28 −24.05 D-2073 22.59 −3.89 21.08 10.82D-2074 21.01 −72.33 3.07 −21.93 D-2075 30.14 −58.62 −3.65 −14.55 D-20765.13 −54.39 17.95 −0.94 D-2077 5.76 −53.36 −6.31 −22.62 D-2078 −2.95−27.45 5.31 −6.53 D-2079 −9.00 −76.93 −1.94 −18.14 D-2080 −20.91 −41.7618.51 2.13 D-2082 1.38 −3.34 −1.76 −12.55 D-2083 13.85 12.36 23.10 10.70D-2084 4.26 −45.51 3.64 −5.08 D-2085 −21.45 −69.45 −1.70 −12.65 D-208614.31 −25.67 5.19 −9.77 D-2087 −10.64 −72.79 11.30 −7.89 D-2088 33.03−49.42 11.55 −4.08 D-2089 −12.31 −71.20 −0.16 −12.87 D-2090 2.23 −13.024.53 −8.06 D-2091 5.50 −30.25 3.26 −8.25 D-2092 25.21 9.46 26.06 9.99D-2093 −30.01 −56.61 −2.51 −10.01 D-2094 3.24 −50.65 12.20 −1.38 D-2096−7.87 −47.43 −6.81 −18.01 D-2097 3.08 −48.50 −3.72 −12.37 D-2098 −5.85−44.10 −15.52 −16.11 D-2099 1.74 −30.12 27.36 8.16 D-2100 −1.85 −27.99−0.85 −15.43 D-2101 9.57 −15.62 −13.37 −32.49 D-2102 17.20 −17.54 8.11−13.47 D-2103 18.26 −14.51 19.92 −2.74 D-2104 −20.60 −52.42 15.88 9.47D-2105 −13.44 −36.40 0.16 −2.69 D-2106 7.45 −24.35 −6.14 −8.94 D-2108−26.79 −47.50 8.03 −6.12 D-2109 28.75 −61.84 7.03 −18.38 D-2110 −15.98−64.33 11.83 −4.94 D-2111 3.58 −25.27 11.63 −0.29 D-2112 −30.49 −70.57−7.11 −23.03 D-2113 −21.34 −45.95 −7.97 −11.53 D-2114 2.37 −38.94 15.524.19 D-2116 −0.77 −16.52 7.68 0.82 D-2117 −34.56 −77.15 −0.97 −27.34D-2118 39.37 1.68 17.34 4.87 D-2119 35.75 −62.09 19.42 −3.86 D-2120−15.98 −37.99 −3.57 −10.72 D-2121 −20.70 −45.64 −0.65 −0.80 D-2122−28.27 −75.45 −8.49 −10.90 D-2123 −3.00 −30.45 −12.77 −21.76 D-2125−20.37 −46.48 12.20 −8.72 D-2126 15.87 −14.95 15.23 −8.82 D-2127 −14.04−41.88 −0.89 −20.13 D-2128 −0.37 −32.88 9.54 6.92 D-2129 −5.66 −42.51−2.74 −16.04 D-2131 −5.23 −60.82 17.74 0.02 D-2132 −7.91 −58.89 14.37−3.66 D-2135 −9.68 −76.64 10.59 1.08 D-2138 32.66 −43.04 15.77 3.66D-2139 −6.65 −37.39 10.37 0.22 D-2140 −12.02 −44.16 2.93 −13.13 D-2141−25.79 −65.81 0.00 −3.03 D-2142 18.26 −9.97 11.72 12.29 D-2143 −40.37−41.64 10.54 6.24 D-2144 17.30 −77.66 −0.73 −30.74 D-2145 15.14 −73.757.55 −20.41 D-2146 −12.43 −66.84 9.71 −17.02 D-2147 −2.79 −80.26 8.20−11.80 D-2148 −13.12 −50.55 6.17 −16.80 D-2149 −14.10 −73.21 1.05 −16.43D-2150 8.57 −11.20 −2.20 −15.48 D-2151 3.25 16.20 −11.67 −16.45 D-2152−27.82 −27.09 3.07 −9.90 D-2153 −8.20 −28.11 14.71 2.34 D-2154 8.26−57.95 5.69 −6.42 D-2155 29.28 −26.90 −0.16 −8.87 D-2156 −8.63 −39.944.29 −1.85 D-2157 40.98 −49.72 15.81 −7.89 D-2158 1.27 −64.90 −20.18−19.77 D-2159 −11.46 −62.75 −5.51 −19.74 D-2160 4.74 −52.84 −2.34 −16.92D-2161 51.01 −43.91 6.03 −11.46 D-2162 −10.09 −45.16 5.61 −2.10 D-21633.12 −48.22 51.05 6.96 D-2164 39.79 −69.13 8.00 −19.97 D-2165 −0.53−74.56 −4.32 −27.16 D-2166 −2.18 −65.81 11.45 −16.77 D-2167 24.40 −53.0727.98 16.39 D-2168 −25.14 −30.88 7.53 5.06 D-2169 7.45 −56.17 16.17 2.89D-2170 −37.06 −62.46 6.69 −14.30 D-2171 0.90 −53.35 −5.11 −15.32 D-217339.14 5.84 12.37 8.02 D-2175 1.55 −63.15 −1.46 −11.53 D-2176 8.07 48.04−6.19 −6.21 D-2177 18.72 22.15 −4.54 −9.19 D-2178 −10.11 0.12 −0.66−4.23 D-2179 0.00 −27.63 1.62 −13.12 D-2180 11.04 13.22 0.17 −3.68D-2181 −28.17 −16.03 2.76 −10.31 D-2182 −25.91 −41.36 0.08 −11.30 D-218324.88 −43.72 −2.67 −10.89 D-2184 18.20 18.68 11.96 11.68 D-2185 −20.32−41.43 −7.54 −26.81 D-2186 −10.37 −73.28 11.38 −12.70 D-2187 19.46−20.05 10.59 10.67 D-2188 1.02 −70.09 −1.74 −15.96 D-2189 −14.50 −38.261.42 −14.19 D-2190 −18.24 −61.71 −9.79 −17.99 D-2191 −15.04 −62.87 15.804.20 D-2193 9.58 −59.04 7.44 −1.72 D-2194 −11.18 −44.01 14.69 −2.00D-2195 19.07 −44.55 15.76 5.22 D-2197 13.12 −25.85 6.61 −12.79 D-2198−4.68 −35.17 4.02 −10.32 D-2199 −8.27 −48.07 14.61 −3.49 D-2201 12.39−68.13 13.26 −8.12 D-2203 5.96 −47.61 22.26 −0.47 D-2204 17.20 −2.7014.12 −7.52 D-2205 −0.37 −2.53 3.93 0.78 D-2206 −1.94 −36.37 16.57 −4.49D-2207 −34.95 −53.37 19.41 −3.59 D-2208 15.05 −61.50 8.37 −14.17 D-220921.64 −73.66 −10.21 −41.26 D-2211 −30.08 −42.14 9.89 −7.13 D-2212 29.46−73.39 7.72 −15.28 D-2213 2.69 −46.37 1.86 8.25 D-2214 38.33 −53.84−5.92 −11.50 D-2215 −23.67 −54.98 17.74 2.12 D-2216 −55.59 −72.78 1.86−11.28 D-2217 29.27 −69.54 14.23 −22.52 D-2218 −26.43 −76.79 −6.39−27.46 D-2219 −10.55 −50.41 7.52 −16.12 D-2220 5.52 −13.82 −3.40 −2.47D-2221 30.40 −61.88 −2.59 −19.75 D-2222 43.30 −48.47 4.32 −3.69 D-222324.63 −62.30 3.32 −5.52 D-2225 −6.63 −42.35 −9.38 −21.15 D-2226 −38.62−62.87 10.46 −8.69 D-2227 −23.94 −50.40 9.29 10.89 D-2228 20.09 −29.7514.14 0.05 D-2229 −18.06 −31.06 11.10 −14.24 D-2231 9.00 −61.35 −13.34−22.09 D-2232 14.77 −76.09 17.15 −3.37 D-2233 7.21 −8.78 0.17 −10.36D-2234 1.28 −72.80 15.23 −43.35 D-2235 −37.16 −74.11 20.67 2.73 D-2237−8.85 −51.03 0.25 −19.94 D-2239 −27.30 −73.35 0.97 −14.70 D-2240 −27.96−75.24 −4.46 −17.10 D-2241 11.91 −72.70 20.45 3.61 D-2242 −0.33 −35.0814.34 7.58 D-2243 −36.81 −74.89 4.31 −11.89 D-2244 30.62 38.57 13.864.28 D-2245 −18.07 −41.66 7.87 −9.64 D-2246 −6.14 56.28 29.46 28.50D-2247 −25.60 −73.58 −4.44 −16.28 D-2248 11.51 −20.86 2.87 −9.83 D-2249−16.91 −39.16 0.34 −14.24 D-2250 15.77 −73.36 10.48 −8.70 D-2251 0.43−79.99 10.74 −17.83 D-2252 6.05 −65.41 5.15 −14.74 D-2253 11.10 −54.8038.74 15.88 D-2254 22.21 −14.24 10.79 −10.26 D-2255 41.07 18.44 6.97−2.34 D-2256 −8.23 −46.19 2.59 3.33 D-2257 −6.65 −9.03 −2.27 −7.54D-2258 −7.71 −76.29 5.44 −7.30 D-2259 14.95 68.74 −17.68 −12.68 D-22609.67 −20.49 0.97 4.43 D-2261 −24.00 −45.64 13.13 −0.23 D-2262 −10.92−17.68 17.57 −0.15 D-2263 3.34 −74.25 4.98 −19.68 D-2264 −8.47 −74.978.13 −7.51 D-2265 −11.80 −58.43 28.74 27.99 D-2268 −4.67 −42.18 −9.56−9.46 D-2269 −8.42 −50.28 5.98 −6.48 D-2270 8.82 −56.73 −3.81 −31.28D-2271 −15.05 −41.32 12.61 5.63 D-2272 −2.18 −51.38 −13.44 −20.07 D-22730.87 7.40 2.43 −10.08 D-2274 6.84 −68.97 −7.29 −31.31 D-2275 17.04−20.30 −4.20 −17.23 D-2276 −19.08 −59.63 8.45 −16.02 D-2277 −5.23 −9.3210.54 8.24 D-2278 16.94 −47.09 5.58 −2.28 D-2279 10.98 −57.75 3.98−13.27 D-2280 10.99 0.50 17.83 8.21 D-2281 3.78 −7.60 7.76 −7.03 D-2282−37.16 −66.11 12.47 −6.97 D-2283 −8.81 −13.66 17.66 −1.44 D-2284 −6.53−56.80 18.51 1.11 D-2285 9.68 −18.56 3.96 −9.57 D-2286 −16.88 −62.0915.77 −11.21 D-2287 −7.59 −35.92 9.08 −6.25 D-2288 4.20 −68.32 −11.91−18.58 D-2289 5.56 −52.09 17.51 4.86 D-2290 47.89 3.79 15.98 −2.50D-2291 4.59 −58.65 −8.12 −6.02 D-2292 5.27 −66.33 −4.90 −20.75 D-229316.75 −52.01 21.10 6.47 D-2295 38.62 12.59 −12.72 −10.00 D-2297 10.8315.03 2.26 6.34 D-2298 −4.69 −2.09 −2.32 −13.00 D-2299 −32.86 −69.774.05 −13.11 D-2302 26.26 −51.02 7.13 4.49 D-2303 11.28 −45.64 −3.77−19.21 D-2305 17.40 −23.21 0.41 −10.26 D-2306 22.94 −53.98 10.63 −13.70D-2307 19.46 −39.96 −1.46 −13.70 D-2308 9.36 −64.54 7.53 −13.04 D-230914.37 −72.06 6.47 −19.58 D-2310 24.72 −29.06 8.38 −9.31 D-2311 −11.17−39.70 8.91 9.42 D-2312 19.21 −71.67 12.28 −11.47 D-2313 19.65 −21.493.15 −11.19 D-2314 26.15 −33.61 14.56 −7.81 D-2315 −13.03 −53.10 10.65−9.69 D-2316 2.42 −29.40 10.91 −11.39 D-2317 −24.49 −67.65 8.33 −5.38D-2318 9.72 −40.13 8.70 −3.99 D-2319 23.72 −77.03 5.51 −63.69 D-232025.70 8.69 8.27 −0.77 D-2321 8.13 −69.42 −17.95 −37.39 D-2323 28.07 2.7220.42 3.41 D-2324 2.89 −56.23 12.76 −8.95 D-2325 13.50 −77.16 −4.23−14.43 D-2326 −3.05 −59.64 21.16 −9.55 D-2327 −12.00 −46.84 6.14 −3.12D-2328 6.84 −40.50 10.62 8.44 D-2329 −1.31 −9.41 7.72 −7.80 D-2330 4.59−69.60 10.79 −8.67 D-2331 −9.54 −73.22 −9.79 −29.41 D-2332 −6.55 −72.418.51 −6.94 D-2333 11.17 −2.82 15.48 11.15 D-2334 −2.66 −75.33 12.72−9.84 D-2335 −23.27 −67.78 10.62 2.39 D-2336 −11.80 40.65 −1.01 −6.78D-2337 −15.32 −76.42 −2.59 −32.42 D-2338 15.49 −74.04 21.13 −1.29 D-233920.66 15.26 −14.02 −26.53 D-2340 26.04 −1.14 7.03 4.64 D-2341 −7.40−74.83 20.50 3.23 D-2342 −15.34 −55.08 4.90 −10.82 D-2343 −12.29 −46.10−10.21 −20.14 D-2344 −23.43 −48.16 4.54 5.25 D-2345 −33.71 −27.73 −9.81−24.68 D-2346 6.88 13.79 2.68 −10.88 D-2347 −18.07 −43.48 20.00 28.83D-2348 −47.43 −50.28 −6.14 −19.13 D-2349 −5.71 −72.76 0.40 −30.56 D-2350−2.48 −3.02 11.13 12.54 D-2351 20.23 −0.55 14.59 16.95 D-2352 −26.04−79.85 2.83 −41.79 D-2353 −26.97 −3.97 11.30 −9.21 D-2355 13.01 −62.857.05 −10.93 D-2356 22.02 −42.24 4.27 −3.90 D-2357 24.78 −73.44 −0.32−16.90 D-2358 28.79 −18.62 1.16 −9.13 D-2359 16.26 −75.14 0.89 −50.36D-2361 42.98 −75.85 2.67 −15.94 D-2363 −8.25 −40.20 −1.94 −1.25 D-236411.83 −29.04 14.99 7.35 D-2365 39.82 −71.52 27.63 −14.32 D-2366 15.7917.44 3.57 −1.02 D-2367 −34.63 −60.76 6.17 −13.96 D-2368 −10.37 −68.3918.24 −21.70 D-2369 −12.78 −49.28 5.09 −0.64 D-2370 0.19 −52.13 −14.96−30.52 D-2371 −21.17 −76.37 −5.27 −26.92 D-2372 −22.46 −77.36 12.13−17.20 D-2373 25.98 −27.45 11.37 −10.77 D-2375 6.51 −70.26 9.54 −30.77D-2376 −18.34 −34.30 7.94 2.16 D-2377 −0.05 −19.93 11.29 −7.45 D-2378−8.76 −55.07 0.08 −17.21 D-2379 −42.39 −67.16 −1.86 −19.30 D-2380 9.85−50.55 6.24 −15.88 D-2381 −18.14 −72.98 28.13 10.21 D-2382 −7.36 −26.0113.66 10.26 D-2383 −39.36 −62.24 18.58 13.73 D-2384 −12.59 −56.52 14.759.42 D-2385 −10.79 −48.93 1.58 −5.69 D-2386 46.35 −44.45 −5.59 −14.76D-2387 −15.88 −46.20 2.10 −19.49 D-2389 13.76 −78.69 −3.01 −28.91 D-23909.10 −30.45 17.67 2.11 D-2391 12.29 −56.75 4.93 −21.32 D-2392 −23.49−74.83 5.02 −33.62 D-2393 16.84 3.98 14.39 1.42 D-2394 −2.71 −70.9813.26 −11.62 D-2395 7.89 −27.03 4.65 2.79 D-2396 16.82 −16.14 16.91−3.49 D-2397 2.12 −35.82 3.89 −3.96 D-2398 9.57 −41.40 18.96 −0.05D-2399 −47.71 −61.72 28.20 0.37 D-2400 −8.35 −72.99 17.07 −18.54 D-2401−28.01 −75.77 1.16 −14.75 D-2402 −33.04 −76.71 18.09 −77.25 D-2404 −7.61−67.29 −1.09 −41.79 D-2405 0.05 −17.64 3.00 −0.09 D-2406 −9.58 −33.64−10.59 −25.33 D-2407 −9.63 −62.24 −0.08 −13.96 D-2408 14.42 −3.00 −6.79−16.32 D-2409 27.04 −34.28 −4.73 −20.25 D-2410 −18.30 −67.46 16.81 −7.29D-2411 −21.28 −57.25 12.72 −8.07 D-2413 4.95 −60.16 10.46 −0.22 D-2414−17.33 −64.07 −14.55 −6.78 D-2415 −15.05 −65.69 −9.46 −17.88 D-2416−0.99 −75.16 11.26 −3.94 D-2417 3.05 −76.41 −0.91 −33.96 D-2418 −1.93−24.85 29.04 17.48 D-2419 −24.40 −47.12 4.18 −3.30 D-2420 16.06 −58.834.35 −25.40 D-2421 −1.37 −74.32 9.56 −21.07 D-2422 −38.14 −48.49 −11.91−24.13 D-2423 −20.41 −60.80 −0.41 −9.66 D-2424 −48.40 −65.04 −2.10 −8.61D-2425 28.94 15.14 14.20 6.12 D-2426 7.89 −47.66 16.10 2.93 D-2427−19.09 −23.01 1.62 −3.94 D-2428 5.56 −11.55 11.62 −5.34 D-2429 0.52−33.21 −11.43 −24.16 D-2430 4.41 −37.30 −3.55 −16.45 D-2431 −37.67−67.95 −1.54 −9.79 D-2432 −25.32 −68.85 −4.13 −20.36 D-2433 0.52 −71.18−7.86 −31.49 D-2434 10.40 −37.19 10.54 −13.54 D-2435 4.86 −57.38 17.3420.49 D-2436 18.88 −35.44 23.12 −12.39 D-2437 25.79 −40.05 15.35 1.03D-2438 15.05 9.99 6.80 −9.53 D-2439 21.63 −41.11 18.17 2.50 D-2440 37.57−0.17 2.20 −12.17 D-2441 −8.06 −54.57 13.61 7.61 D-2442 36.79 57.61 5.61−6.44 D-2443 11.36 −66.78 2.92 −13.62 D-2444 −38.81 −72.85 8.54 −3.71D-2445 −11.04 −64.66 8.08 −2.29 D-2446 3.82 −42.52 −10.45 −14.16 D-2447−7.91 −58.98 13.44 −4.86 D-2448 7.34 −12.66 4.65 −11.66 D-2449 −31.64−2.57 −7.05 −21.21 D-2450 −18.81 −37.87 −0.24 −4.80 D-2451 −9.05 −65.548.96 −5.60 D-2452 7.97 −72.48 −5.11 −13.04 D-2453 −4.77 −45.85 12.38−13.76 D-2454 21.10 −54.04 −4.37 −13.62 D-2455 8.18 −71.54 5.31 −22.07D-2456 11.23 −30.35 −2.67 −15.16 D-2457 5.60 −60.92 1.84 −1.91 D-245821.64 −76.04 4.05 −4.69 D-2459 32.73 −39.55 12.68 2.47 D-2460 −13.78−51.39 10.57 11.22 D-2461 −18.30 −80.09 −13.02 −48.39 D-2463 −24.85−9.05 6.56 −10.92 D-2464 8.42 −30.80 13.50 −8.82 D-2467 −8.91 −35.24−1.05 −16.07 D-2469 9.90 −74.91 12.09 −2.50 D-2470 −16.83 −18.78 9.644.78 D-2471 −13.11 −39.34 17.59 −0.33 D-2472 −51.91 −74.29 0.08 −22.90D-2473 −23.85 −64.30 4.48 −3.65 D-2474 −18.97 −73.74 −9.30 −20.23 D-2476−12.39 −39.15 −5.50 −14.58 D-2477 −36.35 −71.63 −9.00 −8.89 D-2478 5.73−71.48 −7.35 −21.50 D-2479 29.53 −66.03 6.47 −17.15 D-2480 13.11 −73.361.49 −45.78 D-2482 −1.37 −14.22 26.88 7.58 D-2483 −26.46 −76.11 9.13−15.10 D-2484 −7.16 −76.11 14.31 −8.27 D-2485 −6.97 −64.92 −0.65 2.47D-2486 −38.05 −65.90 10.21 −3.14 D-2488 4.40 −65.48 23.24 −1.45 D-248910.61 −50.59 2.76 −16.97 D-2490 −6.92 −56.48 13.36 −5.04 D-2491 −28.11−69.71 10.95 −11.63 D-2492 4.59 34.94 21.34 −5.76 D-2493 9.43 −74.4019.78 −3.04 D-2494 −8.07 −59.67 −2.85 −15.61 D-2495 0.92 −8.01 22.828.68 D-2496 23.04 −8.86 14.79 2.59 D-2497 6.97 −9.77 −5.74 −17.82 D-2498−0.92 −30.75 5.10 −9.22 D-2499 3.63 −39.99 12.48 −5.26 D-2500 −0.4813.29 22.96 11.73 D-2501 −7.36 −19.71 −3.48 −6.31 D-2502 −17.21 −41.5416.86 −1.63

The results from the screening assay of the Tier 2 molecules revealed anadditional 663 potent siRNA molecules that reduced ASGR1 cell surfaceexpression relative to control cells by at least 50% when tested at 5nM. These siRNA molecules were not included in Tier 1 and were notidentified from the bioinformatics analysis of the transcript sequences.At least 263 of these new siRNA molecules reduced ASGR1 cell surfaceexpression relative to control cells by at least 70% and at least 14siRNA molecules reduced ASGR1 cell surface expression relative tocontrol cells by at least 80% when tested at 5 nM.

Example 3. Efficacy of Select ASGR1 siRNA Molecules in RNA FISH Assay

To assess the potency of a subgroup of ASGR1 siRNA molecules in reducingASGR1 expression at the mRNA level, IC50 values were determined for eachsiRNA in an ASGR1 RNA Fluorescence In Situ Hybridization (FISH) assay.Hep3B cells (ATCC HB-8064) were transfected with siRNA usingLipofectamine RNAiMAX transfection reagent (ThermoFisher Scientific13378-150), at 0.035 μL/reaction. Human ASGR1 siRNAs were tested in a 10point dose response format, 3-fold dilutions, ranging from 0-83.3 nM,final concentration. Control siRNAs were tested at 5 nM, finalconcentration and included: a Neutral Control (used for normalization):Non-Targeting RcsC2 (UUACAUCGUUAAUGCGUUA (SEQ ID NO: 4316), an InhibitorPositive Control: human ASGR1 (ACUUCACAGCGAGCACGGA (SEQ ID NO: 4317),and a Transfection Control: human EIF4A3 (GCAUCUUGGUGAAACGUGA (SEQ IDNO: 4318). Cells were seeded over the transfection complex at 2000 cellsper reaction in Perkin Elmer Cell Carrier PDL-coated 384 well assayplates (Perkin Elmer #6007580). The transfection period was 96 hours,after which cells were fixed with 4% methanol free formaldehyde, finalconcentration. Directly post fixation, cells were dehydrated in ethanolfollowing the Dehydrating Cells for Storage or Shipping protocol withinthe manufacturer's protocol for the Affymetrix QuantiGene View RNA HCScreening Assay. Plates were sealed and stored at −20° C.

According to the manufacturer's protocol, cells were rehydrated andprocessed in the Affymetrix QuantiGene View RNA HC Screening Assay, anin situ hybridization method to quantify messenger RNA levels. In thisinstance, the multiplex assay detected human ASGR1 (NM_001671.4; SEQ IDNO: 1), human ASGR2 (NM_0080912.3 or NM_001181.4), and human PPIB(NM_000942.4). The assay was carried out using the QG ViewRNA HCScreening Assay Kit and the QG ViewRNA HC Screening Signal Amp Kit,3-plex (Affymetrix QVP0011 and QVP0213, respectively) and probe sets forthe detection of human ASGR1, ASGR2 and PPIB (Affymetrix, VA6-19401-01,custom type 4 probe, VA1-10148-01 respectively). Each probe set islabeled with a different fluorophore. Protease for the digestion stepwas added at 1:8000, final concentration. Post assay, nuclei andcytoplasm were counterstained using Hoechst 33342 nuclear stain and CellMask Blue reagents (ThermoFisher Scientific H3570 and H32720 at finalconcentrations of 10 ng/μL and 4 ng/μL, respectively). Plate was imagedon the Perkin Elmer Phenix reading Hoechst and Cell Mask Blue in the UVchannel, PPIB/Type1 in the 488 channel, ASGR2/Type4 in the 550 channeland ASGR1/Type6 in the 650 channel. Image acquisition and data analysiswas completed in the Perkin Elmer Columbus software package and wellnormalization/IC50 value generation was completed in Genedata Screener.

The results of the assay are shown in Table 5. The IC50 valuesdetermined in the RNA FISH assay correlate with those determined in theimmunoassay for ASGR1 protein levels described in Example 2. However, atthe time points tested, the IC50 values determined in the RNA FISH assayare higher than those determined in the immunoassay.

TABLE 5 IC50 values determined by RNA FISH assay for select ASGR1 siRNAmolecules Duplex No. IC50 (nM) D-1168 >83.33 D-1170 0.62 D-1171 1.74D-1173 1.65 D-1176 5.12 D-1206 4.70 D-1235 1.57 D-1389 0.68 D-1397 1.06D-1408 >83.33 D-1443 0.58 D-1497 3.65 D-1708 0.05 D-1815 1.18 D-18263.47 D-1981 1.81 D-1989 2.07 D-1999 0.27 D-2000 7.04 D-2142 9.26 D-21434.42 D-2357 0.40 D-2361 1.42 D-2401 1.73 D-2461 4.03

Example 4. Design and Synthesis of Modified ASGR1 siRNA Molecules

To improve the potency and in vivo stability of the ASGR1 siRNAmolecules, chemical modifications were incorporated into a subset of themost potent ASGR1 siRNA molecules from the Tier 1 and Tier 2 screens,including five of the most potent ASGR1 siRNA molecules from the Tier1screen that also had sequence homology with mouse Asgr1 mRNA andcynomolgus monkey ASGR1 mRNA. Specifically, 2′-O-methyl and 2′-fluoromodifications of the ribose sugar were incorporated at specificpositions within the ASGR1 siRNAs. Phosphorothioate internucleotidelinkages were also incorporated at the terminal ends of the antisenseand/or sense sequences. Table 6 below depicts the modifications in thesense and antisense sequences for each of the modified ASGR1 siRNAs. Thenucleotide sequences in Table 6 are listed according to the followingnotations: A, U, G, and C=corresponding ribonucleotide;dT=deoxythymidine; a, u, g, and c=corresponding 2′-O-methylribonucleotide; Af, Uf, Gf, and Cf=corresponding 2′-deoxy-2′-fluoro(“2′-fluoro”) ribonucleotide. Insertion of an “s” in the sequenceindicates that the two adjacent nucleotides are connected by aphosphorothiodiester group (e.g. a phosphorothioate internucleotidelinkage). Unless indicated otherwise, all other nucleotides areconnected by 3′-5′ phosphodiester groups. Each of the siRNA compounds inTable 6 comprises a 19 base pair duplex region with a 2 nucleotideoverhang at the 3′ end of both strands.

TABLE 6 ASGR1 chemically modified siRNA Sequences Duplex SEQ ID NO:SEQ ID NO: No. Sense Sequence (5′-3′) (sense) Antisense Sequence (5′-3′)(antisense) D-3000 AGGcccuAccGcuGGGucudTsdT 3014AGACCcAGCGGuAGGGCCUdTsdT 3665 D-3001 AfgGfcCfcUfAfCfCfGfcUfgGfgUfcUfuUf3015 aGfaCfcCfaGfcgguaGfgGfcCfusUfsu 3666 D-3002AfgGfcCfcUfAfcCfGfcUfgGfgUfcUfuUf 3016 aGfaCfcCfaGfcgGfuaGfgGfcCfusUfsu3667 D-3003 AfgGfcCfcUfacCfGfcUfgGfgUfcUfuUf 3017aGfaCfcCfaGfcgGfUfaGfgGfcCfusUfsu 3668 D-3004AfgGfcCfcUfaCfCfGfCfUfgGfgUfcUfuUf 3018 aGfaCfcCfagcggUfaGfgGfcCfusUfsu3669 D-3005 AfgGfcCfcUfaCfCfgCfUfgGfgUfcUfuUf 3019aGfaCfcCfagCfggUfaGfgGfcCfusUfsu 3670 D-3006AfgGfcCfcUfaCfCfgcUfgGfgUfcUfuUf 3020 aGfaCfcCfaGfCfggUfaGfgGfcCfusUfsu3671 D-3007 AfgGfcCfcuAfCfcGfcUfgGfgUfcUfuUf 3021aGfaCfcCfaGfcGfguAfGfgGfcCfusUfsu 3672 D-3008AfgGfcCfcUfaCfcGfCfugGfgUfcUfuUf 3022 aGfaCfcCfAfgcGfgUfaGfgGfcCfusUfsu3673 D-3009 AfsgsGfcCfcuAfCfcGfcUfgGfgUfcUfuUf 3023asGfsaCfcCfaGfcGfguAfGfgGfcCfusUfsu 3674 D-3010AfgGfcCfcuAfCfcGfcUfgGfgUfcUfuUf 3024 aGfaCfcCfaGfcGfguAfGfgGfcCfuUfu3675 D-3011 AfsgsGfcCfcUfaCfcGfCfugGfgUfcUfuUf 3025asGfsaCfcCfAfgcGfgUfaGfgGfcCfusUfsu 3676 D-3012AfgGfcCfcUfaCfcGfCfugGfgUfcUfuUf 3026 aGfaCfcCfAfgcGfgUfaGfgGfcCfuUfu3677 D-3013 AfgGfcCfCfuaCfcGfcUfgGfgUfcUfuUf 3027aGfaCfcCfaGfcGfgUfAfggGfcCfusUfsu 3678 D-3014AfgGfccCfUfaCfcGfcUfgGfgUfcUfuUf 3028 aGfaCfcCfaGfcGfgUfagGfGfcCfusUfsu3679 D-3015 AfgGfCfccUfaCfcGfcUfgGfgUfcUfuUf 3029aGfaCfcCfaGfcGfgUfaGfGfgcCfusUfsu 3680 D-3016AfggCfCfcUfaCfcGfcUfgGfgUfcUfuUf 3030 aGfaCfcCfaGfcGfgUfaGfggCfCfusUfsu3681 D-3017 AfGfgcCfcUfaCfcGfcUfgGfgUfcUfuUf 3031aGfaCfcCfaGfcGfgUfaGfgGfCfcusUfsu 3682 D-3018aGfGfcCfcUfaCfcGfcUfgGfgUfcUfuUf 3032 aGfaCfcCfaGfcGfgUfaGfgGfccUfsUfsu3683 D-3019 agGfcCfcUfaCfcGfcUfgGfgUfcUfuUf 3033aGfaCfcCfaGfcGfgUfaGfgGfcCfUfsusu 3684 D-3020AfgGfcCfcUfaCfcGfcuGfGfgUfcUfuUf 3034 aGfaCfccAfGfcGfgUfaGfgGfcCfusUfsu3685 D-3021 AfgGfcCfcUfaCfcGfcUfGfggUfcUfuUf 3035aGfaCfCfcaGfcGfgUfaGfgGfcCfusUfsu 3686 D-3022AfgGfcCfcUfaCfcGfcUfggGfUfcUfuUf 3036 aGfacCfCfaGfcGfgUfaGfgGfcCfusUfsu3687 D-3023 AfgGfcCfcUfaCfcGfcUfgGfGfucUfuUf 3037aGfAfccCfaGfcGfgUfaGfgGfcCfusUfsu 3688 D-3024AfgGfcCfcUfaCfcGfcUfgGfguCfUfuUf 3038 agAfCfcCfaGfcGfgUfaGfgGfcCfusUfsu3689 D-3025 AfgGfcCfcUfaCfcGfcUfgGfgUfCfuuUf 3039AfgaCfcCfaGfcGfgUfaGfgGfcCfusUfsu 3690 D-3026AfgGfcCfcUfaCfcGfcUfgGfgUfcuUfUf 3040 AfGfaCfcCfaGfcGfgUfaGfgGfcCfusUfsu3691 D-3027 AfGfgcCfCfuaCfCfgcUfGfggUfCfuuUf 3041AfgaCfCfcaGfCfggUfAfggGfCfcusUfsUf 3692 D-3028agGfCfccUfAfccGfCfugGfGfucUfUfu 3042 aGfAfccCfAfgcGfGfuaGfGfgcCfUfsusu3693 D-3029 AfggCfCfcuAfCfcgCfUfggGfUfcuUfUf 3043AfGfacCfCfagCfGfguAfGfggCfCfususUf 3694 D-3030aGfGfccCfUfacCfGfcuGfGfguCfUfuu 3044 agAfCfccAfGfcgGfUfagGfGfccUfsUfsu3695 D-3031 GuGGGAAGAAAGAuGAAGudTsdT 3045 ACUUcAUCUUUCUUCCcACdTsdT 3696D-3032 GfuGfgGfaAfGfAfAfAfgAfuGfaAfgUfuUf 3046aCfuUfcAfuCfuuucuUfcCfcAfcsUfsu 3697 D-3033GfuGfgGfaAfGfaAfAfgAfuGfaAfgUfuUf 3047 aCfuUfcAfuCfuuUfcuUfcCfcAfcsUfsu3698 D-3034 GfuGfgGfaAfgaAfAfgAfuGfaAfgUfuUf 3048aCfuUfcAfuCfuuUfCfuUfcCfcAfcsUfsu 3699 D-3035GfuGfgGfaAfgAfAfAfGfAfuGfaAfgUfuUf 3049 aCfuUfcAfucuuuCfuUfcCfcAfcsUfsu3700 D-3036 GfuGfgGfaAfgAfAfaGfAfuGfaAfgUfuUf 3050aCfuUfcAfucUfuuCfuUfcCfcAfcsUfsu 3701 D-3037GfuGfgGfaAfgAfAfagAfuGfaAfgUfuUf 3051 aCfuUfcAfuCfUfuuCfuUfcCfcAfcsUfsu3702 D-3038 GfuGfgGfaaGfAfaAfgAfuGfaAfgUfuUf 3052aCfuUfcAfuCfuUfucUfUfcCfcAfcsUfsu 3703 D-3039GfuGfgGfaAfgAfaAfGfauGfaAfgUfuUf 3053 aCfuUfcAfUfcuUfuCfuUfcCfcAfcsUfsu3704 D-3040 GfsusGfgGfaaGfAfaAfgAfuGfaAfgUfuUf 3054asCfsuUfcAfuCfuUfucUfUfcCfcAfcsUfsu 3705 D-3041GfuGfgGfaaGfAfaAfgAfuGfaAfgUfuUf 3055 aCfuUfcAfuCfuUfucUfUfcCfcAfcUfu3706 D-3042 GfsusGfgGfaAfgAfaAfGfauGfaAfgUfuUf 3056asCfsuUfcAfUfcuUfuCfuUfcCfcAfcsUfsu 3707 D-3043GfuGfgGfaAfgAfaAfGfauGfaAfgUfuUf 3057 aCfuUfcAfUfcuUfuCfuUfcCfcAfcUfu3708 D-3044 GfuGfgGfAfagAfaAfgAfuGfaAfgUfuUf 3058aCfuUfcAfuCfuUfuCfUfucCfcAfcsUfsu 3709 D-3045GfuGfggAfAfgAfaAfgAfuGfaAfgUfuUf 3059 aCfuUfcAfuCfuUfuCfuuCfCfcAfcsUfsu3710 D-3046 GfuGfGfgaAfgAfaAfgAfuGfaAfgUfuUf 3060aCfuUfcAfuCfuUfuCfuUfCfccAfcsUfsu 3711 D-3047GfugGfGfaAfgAfaAfgAfuGfaAfgUfuUf 3061 aCfuUfcAfuCfuUfuCfuUfccCfAfcsUfsu3712 D-3048 GfUfggGfaAfgAfaAfgAfuGfaAfgUfuUf 3062aCfuUfcAfuCfuUfuCfuUfcCfCfacsUfsu 3713 D-3049gUfGfgGfaAfgAfaAfgAfuGfaAfgUfuUf 3063 aCfuUfcAfuCfuUfuCfuUfcCfcaCfsUfsu3714 D-3050 guGfgGfaAfgAfaAfgAfuGfaAfgUfuUf 3064aCfuUfcAfuCfuUfuCfuUfcCfcAfCfsusu 3715 D-3051GfuGfgGfaAfgAfaAfgaUfGfaAfgUfuUf 3065 aCfuUfcaUfCfuUfuCfuUfcCfcAfcsUfsu3716 D-3052 GfuGfgGfaAfgAfaAfgAfUfgaAfgUfuUf 3066aCfuUfCfauCfuUfuCfuUfcCfcAfcsUfsu 3717 D-3053GfuGfgGfaAfgAfaAfgAfugAfAfgUfuUf 3067 aCfuuCfAfuCfuUfuCfuUfcCfcAfcsUfsu3718 D-3054 GfuGfgGfaAfgAfaAfgAfuGfAfagUfuUf 3068aCfUfucAfuCfuUfuCfuUfcCfcAfcsUfsu 3719 D-3055GfuGfgGfaAfgAfaAfgAfuGfaaGfUfuUf 3069 acUfUfcAfuCfuUfuCfuUfcCfcAfcsUfsu3720 D-3056 GfuGfgGfaAfgAfaAfgAfuGfaAfGfuuUf 3070AfcuUfcAfuCfuUfuCfuUfcCfcAfcsUfsu 3721 D-3057GfuGfgGfaAfgAfaAfgAfuGfaAfguUfUf 3071 AfCfuUfcAfuCfuUfuCfuUfcCfcAfcsUfsu3722 D-3058 GfUfggGfAfagAfAfagAfUfgaAfGfuuUf 3072AfcuUfCfauCfUfuuCfUfucCfCfacsUfsUf 3723 D-3059guGfGfgaAfGfaaAfGfauGfAfagUfUfu 3073 aCfUfucAfUfcuUfUfcuUfCfccAfCfsusu3724 D-3060 GfugGfGfaaGfAfaaGfAfugAfAfguUfUf 3074AfCfuuCfAfucUfUfucUfUfccCfAfcsusUf 3725 D-3061gUfGfggAfAfgaAfAfgaUfGfaaGfUfuu 3075 acUfUfcaUfCfuuUfCfuuCfCfcaCfsUfsu3726 D-3062 GAGAcGGGcuucAAGAAcudTsdT 3076 AGUUCUUGAAGCCCGUCUCdTsdT 3727D-3063 GfaGfaCfgGfGfCfUfUfcAfaGfaAfcUfuUf 3077aGfuUfcUfuGfaagccCfgUfcUfcsUfsu 3728 D-3064GfaGfaCfgGfGfcUfUfcAfaGfaAfcUfuUf 3078 aGfuUfcUfuGfaaGfccCfgUfcUfcsUfsu3729 D-3065 GfaGfaCfgGfgcUfUfcAfaGfaAfcUfuUf 3079aGfuUfcUfuGfaaGfCfcCfgUfcUfcsUfsu 3730 D-3066GfaGfaCfgGfgCfUfUfCfAfaGfaAfcUfuUf 3080 aGfuUfcUfugaagCfcCfgUfcUfcsUfsu3731 D-3067 GfaGfaCfgGfgCfUfuCfAfaGfaAfcUfuUf 3081aGfuUfcUfugAfagCfcCfgUfcUfcsUfsu 3732 D-3068GfaGfaCfgGfgCfUfucAfaGfaAfcUfuUf 3082 aGfuUfcUfuGfAfagCfcCfgUfcUfcsUfsu3733 D-3069 GfaGfaCfggGfCfuUfcAfaGfaAfcUfuUf 3083aGfuUfcUfuGfaAfgcCfCfgUfcUfcsUfsu 3734 D-3070GfaGfaCfgGfgCfuUfCfaaGfaAfcUfuUf 3084 aGfuUfcUfUfgaAfgCfcCfgUfcUfcsUfsu3735 D-3071 GfsasGfaCfggGfCfuUfcAfaGfaAfcUfuUf 3085asGfsuUfcUfuGfaAfgcCfCfgUfcUfcsUfsu 3736 D-3072GfaGfaCfggGfCfuUfcAfaGfaAfcUfuUf 3086 aGfuUfcUfuGfaAfgcCfCfgUfcUfcUfu3737 D-3073 GfsasGfaCfgGfgCfuUfCfaaGfaAfcUfuUf 3087asGfsuUfcUfUfgaAfgCfcCfgUfcUfcsUfsu 3738 D-3074GfaGfaCfgGfgCfuUfCfaaGfaAfcUfuUf 3088 aGfuUfcUfUfgaAfgCfcCfgUfcUfcUfu3739 D-3075 GfaGfaCfGfggCfuUfcAfaGfaAfcUfuUf 3089aGfuUfcUfuGfaAfgCfCfcgUfcUfcsUfsu 3740 D-3076GfaGfacGfGfgCfuUfcAfaGfaAfcUfuUf 3090 aGfuUfcUfuGfaAfgCfccGfUfcUfcsUfsu3741 D-3077 GfaGfAfcgGfgCfuUfcAfaGfaAfcUfuUf 3091aGfuUfcUfuGfaAfgCfcCfGfucUfcsUfsu 3742 D-3078GfagAfCfgGfgCfuUfcAfaGfaAfcUfuUf 3092 aGfuUfcUfuGfaAfgCfcCfguCfUfcsUfsu3743 D-3079 GfAfgaCfgGfgCfuUfcAfaGfaAfcUfuUf 3093aGfuUfcUfuGfaAfgCfcCfgUfCfucsUfsu 3744 D-3080gAfGfaCfgGfgCfuUfcAfaGfaAfcUfuUf 3094 aGfuUfcUfuGfaAfgCfcCfgUfcuCfsUfsu3745 D-3081 gaGfaCfgGfgCfuUfcAfaGfaAfcUfuUf 3095aGfuUfcUfuGfaAfgCfcCfgUfcUfCfsusu 3746 D-3082GfaGfaCfgGfgCfuUfcaAfGfaAfcUfuUf 3096 aGfuUfcuUfGfaAfgCfcCfgUfcUfcsUfsu3747 D-3083 GfaGfaCfgGfgCfuUfcAfAfgaAfcUfuUf 3097aGfuUfCfuuGfaAfgCfcCfgUfcUfcsUfsu 3748 D-3084GfaGfaCfgGfgCfuUfcAfagAfAfcUfuUf 3098 aGfuuCfUfuGfaAfgCfcCfgUfcUfcsUfsu3749 D-3085 GfaGfaCfgGfgCfuUfcAfaGfAfacUfuUf 3099aGfUfucUfuGfaAfgCfcCfgUfcUfcsUfsu 3750 D-3086GfaGfaCfgGfgCfuUfcAfaGfaaCfUfuUf 3100 agUfUfcUfuGfaAfgCfcCfgUfcUfcsUfsu3751 D-3087 GfaGfaCfgGfgCfuUfcAfaGfaAfCfuuUf 3101AfguUfcUfuGfaAfgCfcCfgUfcUfcsUfsu 3752 D-3088GfaGfaCfgGfgCfuUfcAfaGfaAfcuUfUf 3102 AfGfuUfcUfuGfaAfgCfcCfgUfcUfcsUfsu3753 D-3089 GfAfgaCfGfggCfUfucAfAfgaAfCfuuUf 3103AfguUfCfuuGfAfagCfCfcgUfCfucsUfsUf 3754 D-3090gaGfAfcgGfGfcuUfCfaaGfAfacUfUfu 3104 aGfUfucUfUfgaAfGfccCfGfucUfCfsusu3755 D-3091 GfagAfCfggGfCfuuCfAfagAfAfcuUfUf 3105AfGfuuCfUfugAfAfgcCfCfguCfUfcsusUf 3756 D-3092gAfGfacGfGfgcUfUfcaAfGfaaCfUfuu 3106 agUfUfcuUfGfaaGfCfccGfUfcuCfsUfsu3757 D-3093 GAGcGcAGcuGcuAcuGGudTsdT 3107 ACcAGuAGcAGCUGCGCUCdTsdT 3758D-3094 GfaGfcGfcAfGfCfUfGfcUfaCfuGfgUfuUf 3108aCfcAfgUfaGfcagcuGfcGfcUfcsUfsu 3759 D-3095GfaGfcGfcAfGfcUfGfcUfaCfuGfgUfuUf 3109 aCfcAfgUfaGfcaGfcuGfcGfcUfcsUfsu3760 D-3096 GfaGfcGfcAfgcUfGfcUfaCfuGfgUfuUf 3110aCfcAfgUfaGfcaGfCfuGfcGfcUfcsUfsu 3761 D-3097GfaGfcGfcAfgCfUfGfCfUfaCfuGfgUfuUf 3111 aCfcAfgUfagcagCfuGfcGfcUfcsUfsu3762 D-3098 GfaGfcGfcAfgCfUfgCfUfaCfuGfgUfuUf 3112aCfcAfgUfagCfagCfuGfcGfcUfcsUfsu 3763 D-3099GfaGfcGfcAfgCfUfgcUfaCfuGfgUfuUf 3113 aCfcAfgUfaGfCfagCfuGfcGfcUfcsUfsu3764 D-3100 GfaGfcGfcaGfCfuGfcUfaCfuGfgUfuUf 3114aCfcAfgUfaGfcAfgcUfGfcGfcUfcsUfsu 3765 D-3101GfaGfcGfcAfgCfuGfCfuaCfuGfgUfuUf 3115 aCfcAfgUfAfgcAfgCfuGfcGfcUfcsUfsu3766 D-3102 GfsasGfcGfcaGfCfuGfcUfaCfuGfgUfuUf 3116asCfscAfgUfaGfcAfgcUfGfcGfcUfcsUfsu 3767 D-3103GfaGfcGfcaGfCfuGfcUfaCfuGfgUfuUf 3117 aCfcAfgUfaGfcAfgcUfGfcGfcUfcUfu3768 D-3104 GfsasGfcGfcAfgCfuGfCfuaCfuGfgUfuUf 3118asCfscAfgUfAfgcAfgCfuGfcGfcUfcsUfsu 3769 D-3105GfaGfcGfcAfgCfuGfCfuaCfuGfgUfuUf 3119 aCfcAfgUfAfgcAfgCfuGfcGfcUfcUfu3770 D-3106 GfaGfcGfCfagCfuGfcUfaCfuGfgUfuUf 3120aCfcAfgUfaGfcAfgCfUfgcGfcUfcsUfsu 3771 D-3107GfaGfcgCfAfgCfuGfcUfaCfuGfgUfuUf 3121 aCfcAfgUfaGfcAfgCfugCfGfcUfcsUfsu3772 D-3108 GfaGfCfgcAfgCfuGfcUfaCfuGfgUfuUf 3122aCfcAfgUfaGfcAfgCfuGfCfgcUfcsUfsu 3773 D-3109GfagCfGfcAfgCfuGfcUfaCfuGfgUfuUf 3123 aCfcAfgUfaGfcAfgCfuGfcgCfUfcsUfsu3774 D-3110 GfAfgcGfcAfgCfuGfcUfaCfuGfgUfuUf 3124aCfcAfgUfaGfcAfgCfuGfcGfCfucsUfsu 3775 D-3111gAfGfcGfcAfgCfuGfcUfaCfuGfgUfuUf 3125 aCfcAfgUfaGfcAfgCfuGfcGfcuCfsUfsu3776 D-3112 gaGfcGfcAfgCfuGfcUfaCfuGfgUfuUf 3126aCfcAfgUfaGfcAfgCfuGfcGfcUfCfsusu 3777 D-3113GfaGfcGfcAfgCfuGfcuAfCfuGfgUfuUf 3127 aCfcAfguAfGfcAfgCfuGfcGfcUfcsUfsu3778 D-3114 GfaGfcGfcAfgCfuGfcUfAfcuGfgUfuUf 3128aCfcAfGfuaGfcAfgCfuGfcGfcUfcsUfsu 3779 D-3115GfaGfcGfcAfgCfuGfcUfacUfGfgUfuUf 3129 aCfcaGfUfaGfcAfgCfuGfcGfcUfcsUfsu3780 D-3116 GfaGfcGfcAfgCfuGfcUfaCfUfggUfuUf 3130aCfCfagUfaGfcAfgCfuGfcGfcUfcsUfsu 3781 D-3117GfaGfcGfcAfgCfuGfcUfaCfugGfUfuUf 3131 acCfAfgUfaGfcAfgCfuGfcGfcUfcsUfsu3782 D-3118 GfaGfcGfcAfgCfuGfcUfaCfuGfGfuuUf 3132AfccAfgUfaGfcAfgCfuGfcGfcUfcsUfsu 3783 D-3119GfaGfcGfcAfgCfuGfcUfaCfuGfguUfUf 3133 AfCfcAfgUfaGfcAfgCfuGfcGfcUfcsUfsu3784 D-3120 GfAfgcGfCfagCfUfgcUfAfcuGfGfuuUf 3134AfccAfGfuaGfCfagCfUfgcGfCfucsUfsUf 3785 D-3121gaGfCfgcAfGfcuGfCfuaCfUfggUfUfu 3135 aCfCfagUfAfgcAfGfcuGfCfgcUfCfsusu3786 D-3122 GfagCfGfcaGfCfugCfUfacUfGfguUfUf 3136AfCfcaGfUfagCfAfgcUfGfcgCfUfcsusUf 3787 D-3123gAfGfcgCfAfgcUfGfcuAfCfugGfUfuu 3137 acCfAfguAfGfcaGfCfugCfGfcuCfsUfsu3788 D-3124 GuuGucuGuGuGAucGGAudTsdT 3138 AUCCGAUcAcAcAGAcAACdTsdT 3789D-3125 GfuUfgUfcUfGfUfGfUfgAfuCfgGfaUfuUf 3139aUfcCfgAfuCfacacaGfaCfaAfcsUfsu 3790 D-3126GfuUfgUfcUfGfuGfUfgAfuCfgGfaUfuUf 3140 aUfcCfgAfuCfacAfcaGfaCfaAfcsUfsu3791 D-3127 GfuUfgUfcUfguGfUfgAfuCfgGfaUfuUf 3141aUfcCfgAfuCfacAfCfaGfaCfaAfcsUfsu 3792 D-3128GfuUfgUfcUfgUfGfUfGfAfuCfgGfaUfuUf 3142 aUfcCfgAfucacaCfaGfaCfaAfcsUfsu3793 D-3129 GfuUfgUfcUfgUfGfuGfAfuCfgGfaUfuUf 3143aUfcCfgAfucAfcaCfaGfaCfaAfcsUfsu 3794 D-3130GfuUfgUfcUfgUfGfugAfuCfgGfaUfuUf 3144 aUfcCfgAfuCfAfcaCfaGfaCfaAfcsUfsu3795 D-3131 GfuUfgUfcuGfUfgUfgAfuCfgGfaUfuUf 3145aUfcCfgAfuCfaCfacAfGfaCfaAfcsUfsu 3796 D-3132GfuUfgUfcUfgUfgUfGfauCfgGfaUfuUf 3146 aUfcCfgAfUfcaCfaCfaGfaCfaAfcsUfsu3797 D-3133 GfsusUfgUfcuGfUfgUfgAfuCfgGfaUfuUf 3147asUfscCfgAfuCfaCfacAfGfaCfaAfcsUfsu 3798 D-3134GfuUfgUfcuGfUfgUfgAfuCfgGfaUfuUf 3148 aUfcCfgAfuCfaCfacAfGfaCfaAfcUfu3799 D-3135 GfsusUfgUfcUfgUfgUfGfauCfgGfaUfuUf 3149asUfscCfgAfUfcaCfaCfaGfaCfaAfcsUfsu 3800 D-3136GfuUfgUfcUfgUfgUfGfauCfgGfaUfuUf 3150 aUfcCfgAfUfcaCfaCfaGfaCfaAfcUfu3801 D-3137 GfuUfgUfCfugUfgUfgAfuCfgGfaUfuUf 3151aUfcCfgAfuCfaCfaCfAfgaCfaAfcsUfsu 3802 D-3138GfuUfguCfUfgUfgUfgAfuCfgGfaUfuUf 3152 aUfcCfgAfuCfaCfaCfagAfCfaAfcsUfsu3803 D-3139 GfuUfGfucUfgUfgUfgAfuCfgGfaUfuUf 3153aUfcCfgAfuCfaCfaCfaGfAfcaAfcsUfsu 3804 D-3140GfuuGfUfcUfgUfgUfgAfuCfgGfaUfuUf 3154 aUfcCfgAfuCfaCfaCfaGfacAfAfcsUfsu3805 D-3141 GfUfugUfcUfgUfgUfgAfuCfgGfaUfuUf 3155aUfcCfgAfuCfaCfaCfaGfaCfAfacsUfsu 3806 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D-3622CfgGfaCfuAfCfgAfGfaCfgGfgCfuUfuUf 3636 aAfgCfcCfgUfcuCfguAfgUfcCfgsUfsu4287 D-3623 CfgGfaCfuAfcgAfGfaCfgGfgCfuUfuUf 3637aAfgCfcCfgUfcuCfGfuAfgUfcCfgsUfsu 4288 D-3624CfgGfaCfuAfcGfAfGfAfCfgGfgCfuUfuUf 3638 aAfgCfcCfgucucGfuAfgUfcCfgsUfsu4289 D-3625 CfgGfaCfuAfcGfAfgAfCfgGfgCfuUfuUf 3639aAfgCfcCfguCfucGfuAfgUfcCfgsUfsu 4290 D-3626CfgGfaCfuAfcGfAfgaCfgGfgCfuUfuUf 3640 aAfgCfcCfgUfCfucGfuAfgUfcCfgsUfsu4291 D-3627 CfgGfaCfuaCfGfaGfaCfgGfgCfuUfuUf 3641aAfgCfcCfgUfcUfcgUfAfgUfcCfgsUfsu 4292 D-3628CfgGfaCfuAfcGfaGfAfcgGfgCfuUfuUf 3642 aAfgCfcCfGfucUfcGfuAfgUfcCfgsUfsu4293 D-3629 CfsgsGfaCfuaCfGfaGfaCfgGfgCfuUfuUf 3643asAfsgCfcCfgUfcUfcgUfAfgUfcCfgsUfsu 4294 D-3630CfgGfaCfuaCfGfaGfaCfgGfgCfuUfuUf 3644 aAfgCfcCfgUfcUfcgUfAfgUfcCfgUfu4295 D-3631 CfsgsGfaCfuAfcGfaGfAfcgGfgCfuUfuUf 3645asAfsgCfcCfGfucUfcGfuAfgUfcCfgsUfsu 4296 D-3632CfgGfaCfuAfcGfaGfAfcgGfgCfuUfuUf 3646 aAfgCfcCfGfucUfcGfuAfgUfcCfgUfu4297 D-3633 CfgGfaCfUfacGfaGfaCfgGfgCfuUfuUf 3647aAfgCfcCfgUfcUfcGfUfagUfcCfgsUfsu 4298 D-3634CfgGfacUfAfcGfaGfaCfgGfgCfuUfuUf 3648 aAfgCfcCfgUfcUfcGfuaGfUfcCfgsUfsu4299 D-3635 CfgGfAfcuAfcGfaGfaCfgGfgCfuUfuUf 3649aAfgCfcCfgUfcUfcGfuAfGfucCfgsUfsu 4300 D-3636CfggAfCfuAfcGfaGfaCfgGfgCfuUfuUf 3650 aAfgCfcCfgUfcUfcGfuAfguCfCfgsUfsu4301 D-3637 CfGfgaCfuAfcGfaGfaCfgGfgCfuUfuUf 3651aAfgCfcCfgUfcUfcGfuAfgUfCfcgsUfsu 4302 D-3638cGfGfaCfuAfcGfaGfaCfgGfgCfuUfuUf 3652 aAfgCfcCfgUfcUfcGfuAfgUfccGfsUfsu4303 D-3639 cgGfaCfuAfcGfaGfaCfgGfgCfuUfuUf 3653aAfgCfcCfgUfcUfcGfuAfgUfcCfGfsusu 4304 D-3640CfgGfaCfuAfcGfaGfacGfGfgCfuUfuUf 3654 aAfgCfccGfUfcUfcGfuAfgUfcCfgsUfsu4305 D-3641 CfgGfaCfuAfcGfaGfaCfGfggCfuUfuUf 3655aAfgCfCfcgUfcUfcGfuAfgUfcCfgsUfsu 4306 D-3642CfgGfaCfuAfcGfaGfaCfggGfCfuUfuUf 3656 aAfgcCfCfgUfcUfcGfuAfgUfcCfgsUfsu4307 D-3643 CfgGfaCfuAfcGfaGfaCfgGfGfcuUfuUf 3657aAfGfccCfgUfcUfcGfuAfgUfcCfgsUfsu 4308 D-3644CfgGfaCfuAfcGfaGfaCfgGfgcUfUfuUf 3658 aaGfCfcCfgUfcUfcGfuAfgUfcCfgsUfsu4309 D-3645 CfgGfaCfuAfcGfaGfaCfgGfgCfUfuuUf 3659AfagCfcCfgUfcUfcGfuAfgUfcCfgsUfsu 4310 D-3646CfgGfaCfuAfcGfaGfaCfgGfgCfuuUfUf 3660 AfAfgCfcCfgUfcUfcGfuAfgUfcCfgsUfsu4311 D-3647 CfGfgaCfUfacGfAfgaCfGfggCfUfuuUf 3661AfagCfCfcgUfCfucGfUfagUfCfcgsUfsUf 4312 D-3648cgGfAfcuAfCfgaGfAfcgGfGfcuUfUfu 3662 aAfGfccCfGfucUfCfguAfGfucCfGfsusu4313 D-3649 CfggAfCfuaCfGfagAfCfggGfCfuuUfUf 3663AfAfgcCfCfguCfUfcgUfAfguCfCfgsusUf 4314 D-3650cGfGfacUfAfcgAfGfacGfGfgcUfUfuu 3664 aaGfCfccGfUfcuCfGfuaGfUfccGfsUfsu4315

Synthesis of chemically modified siRNA sequences was performed on the GEAKTA OligoPilot 100.

Materials:

Acetonitrile (DNA Synthesis Grade, AXO152-2505, EMD)

Capping Reagent A (20% N-methylimidazole in acetonitrile, BI0224-0505,EMD, Lot #56090)

Capping Reagent B1 (20% acetic anhydride in acetonitrile, BI0347-0505,EMD, Lot #55015)

Capping Reagent B2 (30% 2,6-lutidine in aceotnitrile, BI0349-0505, EMD,Lot #55176)

Capping Reagent B1 and B2 were mixed together 1:1 (v/v).

Activator (0.3 M benzylthiotetrazole (BTT) in acetonitrile, BI0166-1005,EMD Lot #55106, over molecular sieves)

Detritylation Reagent (3% dichloroacetic acid in toluene, BI0832-2505,EMD, Lot #55316)

Oxidation Reagent (0.05 M iodine in 90:10 pyridine/water, BI0424-1005,EMD, Lot #54323)

Diethylamine solution (20% DEA in acetonitrile, NC0017-0505, EMD, Lot#55202)

Ammonium hydroxide (concentrated, J. T. Baker)

Thiolation Reagent, 0.2 M phenylacetic disulfide (PADS, Aldrich) in50:50 2-methylpyridine (picoline, Aldrich)/N-methylpyrrolidinone (NMP),Aldrich)

Thymidine (Thermo Fisher Scientific) and 2′-O-methyl and 2′-fluorophosphoramidites of adenosine, guanosine, cytosine, and uridine (ThermoFisher Scientific), 0.15 M in acetonitrile over ˜10 mL of molecularsieves (J. T. Baker)

Primer Support 5G UnyLinker 350, Lot #10236161, 343 μmol/g, 0.60 g (206μmol) or Primer Support 5G Amino with GalNAc cluster

Synthesis:

Reagent solutions, phosphoramidite solutions, and solvents were attachedto the instrument. Solid support was added to the column (6.3 mL), andthe column was affixed to the instrument. The column was flushed withacetonitrile. The synthesis was started using the Unicorn software. Thephosphoramidite and reagent solution lines were purged. The synthesiswas accomplished by repetition of thedeprotection/coupling/oxidation/capping synthesis cycle. To the solidsupport was added detritylation reagent to remove the 5′-dimethoxytrityl(DMT) protecting group. The solid support was washed with acetonitrile.To the support was added phosphoramidite and activator solution followedby recycling to couple the incoming nucleotide to the free 5′-hydroxylgroup. The support was washed with acetonitrile. To the support wasadded oxidation or thiolation reagent to convert the phosphite triesterto the phosphate triester or phosphorothioate. To the support was addedcapping reagents A and B to terminate any unreacted oligonucleotidechains. The support was washed with acetonitrile. After the finalreaction cycle, the resin was washed with diethylamine solution toremove the 2-cyanoethyl protecting groups. The support was washed withacetonitrile.

Cleavage:

The synthesis column was removed from the synthesizer and dried undervacuum for 20 minutes. The column was opened, and the solid support wastransferred to a 100 mL bottle. To the solid support was added 40 mL ofconcentrated ammonium hydroxide. The cap was tightly affixed to thebottle, and the mixture was heated at 65° C. overnight. The bottle wasmoved to the freezer and cooled for 20 minutes before opening in thehood. The mixture was filtered through a 60 mL M fritted glass funnel.The bottle and solid support were rinsed with 20 mL of 50:50ethanol/water and then 40 mL of water.

Analysis and Purification:

A portion of the combined filtrate was analyzed and purified by anionexchange chromatography. The pooled fractions were desalted by sizeexclusion chromatography and analyzed by ion pair-reversed phase HPLC.The pooled fractions were lyophilized to obtain a white amorphouspowder.

Analytical Anion Exchange Chromatography (AEX):

Column: Thermo DNAPac PA200RS (4.6×50 mm, 4 μm)

Instrument: Agilent 1100 HPLC

Buffer A: 20 mM sodium phosphate, 10% acetonitrile, pH 8.5

Buffer B: 20 mM sodium phosphate, 10% acetonitrile, pH 8.5, 1 M sodiumbromide

Flow rate: 1 mL/min at 40° C.

Gradient: 20-65% B in 6.2 min

Preparative anion exchange chromatography (AEX):

Column: Tosoh TSK Gel SuperQ-SPW, 21×150 mm, 13 μm

Instrument: Agilent 1200 HPLC

Buffer A: 20 mM sodium phosphate, 10% acetonitrile, pH 8.5

Buffer B: 20 mM sodium phosphate, 10% acetonitrile, pH 8.5, 1 M sodiumbromide

Flow rate: 8 mL/min

Injection volume: 5 mL

Gradient: 35-55% B over 20 min

Preparative Size Exclusion Chromatography (SEC):

Column: GE Hi-Prep 26/10

Instrument: GE AKTA Pure

Buffer: 20% ethanol in water

Flow Rate: 10 mL/min

Injection volume: 15 mL using sample loading pump

Ion Pair-Reversed Phase (IP-RP) HPLC:

Column: Water Xbridge BEH OST C18, 2.5 μm, 2.1×50 mm

Instrument: Agilent 1100 HPLC

Buffer A: 15.7 mM DIEA, 50 mM HFIP in water

Buffer B: 15.7 mM DIEA, 50 mM HFIP in 50:50 water/acetonitrile

Flow rate: 0.5 mL/min

Gradient: 10-30% B over 6 min

Annealing:

A small amount of the sense strand and the antisense strand were weighedinto individual vials. To the vials was added siRNA reconstitutionbuffer (Qiagen) to an approximate concentration of 2 mM based on the dryweight. The actual sample concentration was measured on the NanoDrop One(ssDNA, extinction coefficient=33 μg/OD260). The two strands were thenmixed in an equimolar ratio, and the sample was heated for 3 minutes ina 90° C. water bath and allowed to cool slowly to room temperature. Thesample was analyzed by AEX. The RNA duplex was observed to have a longerretention time by analytical AEX than the single strands. The duplex wasregistered and submitted for in vitro (see methods described in Examples2 and 3) and in vivo (see methods described in Example 6) testing.

Example 5. Synthesis of GalNAc-Containing Ligand

This example describes the synthesis of a tetravalent GalNAc moiety,which can be conjugated to the double-stranded RNA molecules in the RNAiconstructs of the invention to facilitate delivery and uptake of theRNAi constructs by the liver (e.g. hepatocytes). The synthetic scheme isdepicted in FIG. 5.

Resin-Bound Tetraantennary GalNAc Step 1:(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(trityloxy)propoxy)-4-oxobutanoicacid (1)

The (R)-(9H-fluoren-9-yl)methyl(1-hydroxy-3-(trityloxy)propan-2-yl)carbamate (20 g, 36.0 mmol),succinic anhydride (7.20 g, 72 mmol), polystyrene-supported DMAP (3mmol/g, 24 g, 72 mmol) and triethylamine (10 mL, 72 mmol) were taken upin DCM (720 mL). The suspension was stirred at room temperature for 16h. The reaction was filtered through Celite (to remove PS-dmap), and thefilter cake was rinsed with DCM (200 mL). The combined filtrate wasextracted with saturated aqueous NaCl (3×100 mL). The organic layer wasdried over sodium sulfate and concentrated to afford the crude titlecompound (23.6 g, 36.0 mmol, 100% yield) which was used in the next stepwithout further purification. MS m/z 678.2 (M+Na).

Step 2

In a 50-mL conical tube, the activated hemisuccinate was prepared asfollows:(S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(trityloxy)propoxy)-4-oxobutanoicacid (0.5 g, 0.763 mmol) and TATU (344 mg, 1.07 mmol) were dissolved in5 mL of DMF, and the tube was swirled for 3 minutes. Hunig's base (0.400mL, 2.29 mmol) was added. Meanwhile, the resin (Primer Support 5G Aminofrom GE Lifesciences, 0.46 mmol/g, 1.66 g, 0.763 mmol) was swelled in a50-mL falcon tube in 10 mL of DMF. The activated hemisuccinate solutionwas added. The tube was gently shaken at room temperature at 400 rpm.The reaction mixture was filtered, then rinsed with DCM (50 mL), 10%MeOH-DCM (50 mL), then DCM (50 mL) and dried under vacuum. The resin wascapped by adding a solution of acetic anhydride (5.625 mL, 59 mmol),pyridine (16.65 mL) and triethylamine (0.225 mL), and shaking at 400 rpmfor 2 h at room temperature. The resin was filtered, rinsed with DCM (50mL), 10% MeOH-DCM (50 mL), and DCM (50 mL) and dried under vacuum, toafford Intermediate 2 (2.55 g, 0.255 mmol/g, 0.65 mmol), which was usedas is in the next step.

Step 3

Intermediate 2 (2.55 g, 0.65 mmol) was suspended in a solution of 20%4-methylpiperidine in DMF (15 mL) and stirred for 5 minutes. Thesolution was drained, and the process was repeated two more times toafford the deprotected intermediate. An activated solution ofFmoc-protected 6-aminohexanoic acid was made by dissolvingFmoc-protected 6-aminohexanoic acid (1.41 g, 4.0 mmol) and TATU (1.288g, 4.0 mmol) in DMF (10 mL). After 5 minutes, Hunig's base (1.05 mL,6.05 mmol) was added. This solution was added to the deprotected resin.The mixture was shaken at 400 rpm at room temperature overnight. Thereaction was filtered, and the resin was washed with DMF (3×30 mL). Thesame procedure (deprotection, preparation of the activated acid, andcoupling) was repeated to afford crude Intermediate 3 (2.40 g, 0.184mmol/g, 0.442 mmol), which was used in the next step.

Step 4

Intermediate 3 was suspended in a solution of 20% 4-methylpiperidine inDMF (15 mL) and stirred for 5 minutes. The solution was drained, and theprocess was repeated two more times to afford the deprotectedintermediate. An activated solution of bis-Fmoc-protected lysine wasprepared by dissolving bis-Fmoc-protected lysine (2.07 g, 3.5 mmol) andTATU (1.13 g, 3.5 mmol) in DMF (10 mL) and stirring for 5 minutes.Hunig's base (0.96 mL, 5.5 mmol) was added. This solution was added tothe deprotected resin. The suspension was shaken at 400 rpm at roomtemperature overnight. The reaction mixture was filtered, and the resinwas then washed with DMF (3×30 mL). The resin was deprotected using theprocedure above. An activated solution of bis-Fmoc-protected lysine wasprepared as above, except that the amount of bis-Fmoc-protected lysinewas 2.96 g (5.0 mmol), the amount of TATU was 1.61 g (5.0 mmol), and theamount of Hunig's base was 1.31 mL (7.5 mmol), and the deprotected resinwas coupled to the activated acid by shaking at 400 rpm at roomtemperature overnight. The resin was washed with DMF (3×30 mL) and thenDCM (3×30 mL), and dried to afford crude Intermediate 4 (2.28 g, 0.165mmol/g, 0.376 mmol).

Step 5

Intermediate 4 (0.4 mmol) was suspended in a solution of 20%4-methylpiperidine in DMF (25 mL) and stirred for 5 minutes. Thesolution was drained, and the process was repeated one more time toafford the deprotected intermediate. To a solution of5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoicacid (5, 2.68 g, 6 mmol) in DMF (20 mL) was added TATU (1.92 g, 6.0mmol) and the solution was stirred for 5 min. Hunig's base (1.57 mL, 9.0mmol) was added to the solution and the mixture was then added to thedeprotected intermediate. The suspension was kept at room temperatureovernight and the solvent was drained. The resin was washed with DMF(3×30 mL) and DCM (3×30 mL). The resin was treated with 3%Dichloroacetic acid in Tol with 5% TIPS (25 mL) and after 5 min thesolvent was drained. The process was repeated two more times to giveintermediate 6, which was used in the next step directly (e.g.conjugation reaction to 5′ or 3′ end of sense strand of an RNAiconstruct of the invention).

The chemically modified ASGR1 siRNAs alone or conjugated to the GalNAcmoiety are evaluated for efficacy in reducing ASGR1 expression by the invitro immunoassay described in Example 2, the RNA FISH assay describedin Example 3, or the in vivo mouse model described in Example 6.

Example 6. In Vivo Efficacy of ASGR1 siRNA Molecules

To assess the efficacy of chemically modified ASGR1 siRNA molecules inreducing ASGR1 liver expression in vivo, the modified ASGR1 siRNAmolecules (complexed with Invivofectamine® reagent) or GalNAc-siRNAconjugates are administered to C57BL/6J mice intravenously orsubcutaneously. Specifically, mice are injected with buffer, indicatedsiRNA and matched control siRNA at 1-5 mg/kg body weight in 0.25 mlbuffer on day 0. Animals are harvested for further analysis at day 2,day 4 and day 7. Liver total RNA from harvested animals is processed forqPCR analysis. The efficacy of the ASGR1 siRNA is assessed by comparingthe amount of Asgr1 mRNA and ASGR1 protein in liver tissue of thesiRNA-treated animals to the amount of Asgr1 mRNA and ASGR1 protein inliver tissue of animals injected with buffer or control siRNAs.

Serum levels of alkaline phosphatase and LDL cholesterol may also bemeasured in the mice at various times following injection with ASGR1siRNA molecules or matched controls to assess the in vivo efficacy ofASGR1 siRNA molecules. Elevated serum alkaline phosphatase levelscorrelate with reduced serum levels of non-HDL cholesterol and reducedrisk of coronary artery disease (Nioi et al., New England Journal ofMedicine, Vol. 374(22):2131-2141, 2016, which is hereby incorporated byreference in its entirety). Thus, serum alkaline phosphatase levels canbe used as a surrogate biomarker of efficacy of a particular ASGR1 siRNAto reduce serum non-HDL cholesterol levels or risk of coronary arterydisease. Efficacious ASGR1 siRNA molecules are those that producereduced serum non-HDL cholesterol (e.g. LDL cholesterol) levels orincreased serum alkaline phosphatase levels in treated animals ascompared to the levels in animals injected with buffer or controlsiRNAs.

Example 7. In Vitro Efficacy of ASGR1 Chemically Modified siRNAMolecules

Select chemically modified siRNA molecules listed in Table 6 wereconjugated to the triantennary GalNAc moiety shown in Formula VII, thestructure of which is reproduced below. The GalNAc moiety was conjugatedto the 3′ end of the sense strand of each duplex through aphosphodiester linkage.

The GalNAc-siRNA conjugates were evaluated for their ability to inhibitASGR1 expression in the Hep3B cell transfection assay and human ASGR1CHO cell free uptake assay. The Hep3B cell transfection immunoassay isdescribed in Example 2 above. The free uptake assay utilizing ChineseHamster Ovary (CHO) cells stably expressing human ASGR1 was conducted asfollows. GalNAc-conjugated siRNA molecules in F-12K media (CorningCellgro #10-025-CV) were prepared in 384-well plates. CHO cells stablyexpressing human ASGR1 in F-12K media supplemented with 10% fetal bovineserum and 1% antibiotic/antimycotic were added to each well. Cells wereincubated for 4 days at 37° C. and 5% CO₂. Four days after siRNAdelivery, cells were fixed in formaldehyde, blocked with bovine serumalbumin, and subsequently stained with an anti-ASGR1 primary antibody(Amgen clone 7E11, light and heavy chain sequences provided in SEQ IDNOs: 3 and 4, respectively) for either 1 hour at room temperature orovernight at 4° C. Plates were washed three times with phosphatebuffered saline (PBS). Cells were then incubated in the dark for 45minutes at room temperature with Alexa488-conjugated anti-human IgGsecondary antibody and nuclear stain Hoechst 33342 (Invitrogen #H3570)to assess cell number. Following three PBS washes, the plates wereimaged on an Opera Phenix high-content screening system (PerkinElmer)using 488/500-550 and 375/435-480 excitation/emission filter settings tomeasure anti-ASGR1 antibody staining and nuclear staining, respectively.Data were analyzed using Columbus image analysis software and GeneDataScreener software to quantify several measures of ASGR1 protein levels,cell count, and cell morphology on a per cell and per well basis.

The GalNAc-siRNA conjugates were tested at twenty-two different dosesranging from 0.000012 to 25 μM in each of the assays and dose-responsecurves were constructed. IC50 values and maximum antagonist activityvalues (relative to control cells; −1.0 max antagonist activityrepresents complete inhibition) were calculated from the dose-responsecurves. The results of the assays are shown in Table 7 below.

TABLE 7 In vitro efficacy of GalNAc-ASGR1 siRNA conjugates Hep3B hASGR1CHO Hep3B Transfected hASGR1 CHO Free Uptake Transfected Max AntagonistFree Uptake Max Antagonist Duplex No. IC50 (μM) Activity IC50 (μM)Activity D-3033 0.00325 −0.84 >0.5 −0.34 D-3034 >0.167 −0.46 >0.5 −0.08D-3036 0.00355 −0.97 >0.5 −0.48 D-3037 0.00395 −0.96 >0.5 −0.55D-3044 >0.5 −0.05 >0.5 0.13 D-3046 0.00481 −0.82 >0.5 −0.14 D-30480.00288 −0.93 >0.5 −0.31 D-3050 0.00386 −0.99 >0.5 −0.36 D-3051 0.0124−0.82 >0.5 −0.51 D-3053 0.00349 −0.84 >0.5 −0.48 D-3055 0.00346−0.82 >0.5 −0.26 D-3057 0.0021 −0.86 >0.5 −0.58 D-3058 >0.5 0.04 >0.50.26 D-3059 0.00966 −0.82 >0.5 −0.12 D-3060 0.00527 −0.71 >0.5 −0.06D-3061 >0.5 0.14 >0.5 −0.20 D-3063 0.00541 −1.0 0.395 −0.20 D-30710.00435 −0.9 >12.5 −0.10 D-3094 0.00544 −0.9 >25.0 −0.40 D-3102 0.00649−0.9 >12.5 −0.10 D-3125 0.00144 −0.9 0.694 −0.40 D-3133 0.00245−0.8 >12.5 −0.20 D-3156 0.00206 −0.5 >25.0 −0.10 D-3164 1.17 −0.4 2.14−0.10 D-3187 0.00653 −0.6 >25.0 −0.10 D-3195 >0.0977 −0.5 0.179 −0.10D-3219 >0.5 0.08 >0.5 −0.09 D-3220 >0.5 0.12 >0.5 −0.11 D-3222 0.00406−0.95 >0.5 −0.14 D-3223 0.00818 −0.74 >0.5 −0.07 D-3228 0.000479−0.9 >25.0 −0.20 D-3230 >12.5 −0.2 >25.0 −0.10 D-3231 >12.5 −0.2 >25.0−0.20 D-3232 0.30214 −0.6 >25.0 −0.10 D-3233 0.00468 −0.4 >25.0 −0.10D-3234 1.5879 −0.8 >25.0 0.20 D-3235 0.00109 −0.9 >25.0 0.10 D-32360.0047 −0.7 >25.0 −0.20 D-3237 0.000178 −0.9 >25.0 −0.20 D-3238 0.00336−0.9 >25.0 −0.30 D-3239 0.00394 −0.9 >25.0 −0.20 D-3240 0.00458−0.9 >25.0 −0.20 D-3241 >12.5 −0.2 >25.0 −0.10 D-3242 >12.5 −0.2 >25.00.20 D-3243 0.00122 −0.9 >25.0 −0.10 D-3244 >12.5 −0.1 >25.0 0.10D-3245 >12.5 −0.4 >25.0 0.10 D-3246 >12.5 −0.2 >25.0 −0.20 D-3247 >12.5−0.2 >25.0 −0.10 D-3249 0.0137 −0.6 >25.0 0.00 D-3257 2.33 −0.4 >12.5−0.30 D-3280 0.00407 −1.0 >25.0 −0.10 D-3288 0.011 −0.7 >12.5 0.10D-3311 0.0068 −0.8 >25.0 −0.20 D-3319 >12.5 −0.3 >12.5 0.00 D-33420.00217 −0.9 0.703 −0.30 D-3350 0.00512 −0.7 >12.5 −0.10 D-3373 0.00961−0.6 >25.0 0.00 D-3381 >12.5 −0.2 >12.5 −0.20 D-3404 0.00497 −0.8 >25.0−0.30 D-3412 0.00915 −0.6 >12.5 −0.10 D-3435 0.00302 −0.7 >25.0 0.10D-3443 >12.5 −0.3 >6.25 −0.20 D-3467 0.00473 −0.7 >25.0 −0.20D-3468 >12.5 −0.2 >25.0 0.20 D-3470 0.079127 −0.9 >25.0 −0.30 D-34710.38449 −0.9 >25.0 −0.10 D-3474 0.648 −0.4 >25.0 0.00 D-3476 0.23421−0.9 >25.0 −0.40 D-3478 >12.5 −0.1 >25.0 0.20 D-3479 0.948 −0.3 >25.0−0.10 D-3480 0.274 −0.5 >25.0 0.10 D-3481 0.00181 −0.8 >25.0 0.00 D-34820.54988 −0.9 >25.0 −0.10 D-3483 0.35517 −0.9 >25.0 −0.20 D-3484 1.3218−0.9 >25.0 −0.20 D-3485 0.00376 −0.9 1.71 −0.30 D-3486 0.0054 −0.9 >25.0−0.10 D-3487 0.00419 −0.8 >25.0 −0.20 D-3488 0.00582 −0.9 >25.0 −0.10D-3489 0.737 −0.3 >25.0 0.10 D-3490 >12.5 0.0 >25.0 −0.10 D-3491 0.00799−0.8 2.53 −0.30 D-3505 >12.5 −0.3 11 −0.30 D-3528 0.000536 −1.0 >25.0−0.10 D-3536 0.00559 −0.8 >12.5 0.10 D-3559 0.00285 −1.0 >25.0 0.00D-3567 0.0027 −1.0 >25.0 −0.20

Several of the GalNAc-siRNA conjugates knocked down ASGR1 expression bygreater than 80% when transfected into Hep3B cells. In particularGalNAc-siRNA conjugates targeting nucleotides 692 to 710 of the humanASGR1 transcript variant 1 (NM_001671.4; SEQ ID NO: 1) having variouschemical modification patterns (e.g. Duplex Nos. 3036, 3037, 3051, 3053,and 3057) exhibited low nanomolar IC50 values when transfected intoHep3B cells and about 50% maximum knockdown activity in the CHO cellfree uptake assay. Changing the GalNAc moiety in the conjugates to othertriantennary GalNAc structures described herein (e.g. Formula XVI)improved performance of the GalNAc-siRNA conjugates in the free uptakeassay (data not shown).

Example 8. Design and Efficacy of Additional GalNAc-ASGR1 siRNAMolecules

Additional GalNAc-ASGR1 siRNA conjugates were made that had varyingpatterns of chemical modifications, different sequences, and differentGalNAc moieties. Table 8 below lists the modifications in the sense andantisense sequences and the structure and site of conjugation for theGalNAc moiety for each of the GalNAc-ASGR1 siRNA conjugates. Thenucleotide sequences in Table 8 are listed according to the followingnotations: A, U, G, and C=corresponding ribonucleotide; dT, dA, dG,dC=corresponding deoxyribonucleotide; a, u, g, and c=corresponding2′-O-methyl ribonucleotide; Af, Uf, Gf, and Cf=corresponding2′-deoxy-2′-fluoro (“2′-fluoro”) ribonucleotide; Phos=terminalnucleotide has a monophosphate group at its 5′ end; and invAb=invertedabasic nucleotide (i.e. abasic nucleotide linked to adjacent nucleotidevia a substitutent at its 3′ position (a 3′-3′ linkage)). Insertion ofan “s” in the sequence indicates that the two adjacent nucleotides areconnected by a phosphorothiodiester group (e.g. a phosphorothioateinternucleotide linkage). Unless indicated otherwise, all othernucleotides are connected by 3′-5′ phosphodiester groups. GalNAcstructures are shown in the referenced formulas, which are depictedabove.

TABLE 8 GalNAc-ASGR1 siRNA Conjugates Site of GalNAc SEQ SEQ DuplexGalNAc Moiety ID ID No. Moiety Conjug. Sense Sequence (5′-3′) NO:Antisense Sequence (5′-3′) NO: D-3651 Formula 3′ end{Phos}GfsusGfgGfaAfGfAfAfdAgAfuGfaAfgUfuUf 4319{Phos}asCfsuUfcAfuCfuuucuUfcCfcAfcsUfsu 4513 VII of sense strand D-3652Formula 3′ end {Phos}GfsusGfgGfaAfGfdAAfAfgAfuGfaAfgUfuUf 4320{Phos}asCfsuUfcAfuCfuuucuUfcCfcAfcsUfsu 4513 VII of sense strand D-3653Formula 3′ end {Phos}GfsusGfgGfaAfgdAAfAfGfAfuGfaAfgUfuUf 4321{Phos}asCfsuUfcAfucuuuCfuUfcCfcAfcsUfsu 4514 VII of sense strand D-3654Formula 3′ end {Phos}GfsusGfgGfaAfgAfAfdAGfAfuGfaAfgUfuUf 4322{Phos}asCfsuUfcAfucuuuCfuUfcCfcAfcsUfsu 4514 VII of sense strand D-3655Formula 3′ end {Phos}GfsusGfgGfaaGfAfadAgAfuGfaAfgUfuUf 4323{Phos}asCfsuUfcAfuCfuUfucUfUfcCfcAfcsUfsu 4515 VII of sense strandD-3656 Formula 3′ end {Phos}GfsusGfgGfaaGfAfdAAfgAfuGfaAfgUfuUf 4324{Phos}asCfsuUfcAfuCfuUfucUfUfcCfcAfcsUfsu 4515 VII of sense strandD-3657 Formula 3′ end {Phos}GfsusGfgGfaaGfdAaAfgAfuGfaAfgUfuUf 4325{Phos}asCfsuUfcAfuCfuUfucUfUfcCfcAfcsUfsu 4515 VII of sense strandD-3658 Formula 3′ end {Phos}gsusgggaAfgAfadAgaugaaguuu 4326{Phos}asCfsuUfcAfuCfuUfuCfuUfcCfcacsusu 4516 VII of sense strand D-3659Formula 3′ end {Phos}GfsusGfgGfaAfGfAfAfAfgAfuGfaAfguuu 4327{Phos}asCfsuUfcAfuCfuuucuUfcCfcAfcsUfsu 4513 VII of sense strand D-3660Formula 3′ end {Phos}GfscsAfgCfuGfcUfaCfuGfgUfuCfuCfuUf 4328{Phos}gsAfsgAfaCfcAfgUfaGfcAfgCfuGfcsUfsu 4517 VII of sense strandD-3661 Formula 3′ end {Phos}GfscsAfgCfugCfUfAfCfuGfgUfuCfuCfuUf 4329{Phos}gsAfsgAfaCfcAfguagCfAfgCfuGfcsUfsu 4518 VII of sense strand D-3662Formula 3′ end {Phos}gsCfsaGfcUfgCfUfAfCfuGfgUfuCfuCfuUf 4330{Phos}gsAfsgAfaCfcAfguagCfaGfcUfgCfsusUf 4519 VII of sense strand D-3663Formula 3′ end {Phos}GfscsAfgCfuGfcUfAfCfUfggUfuCfuCfuUf 4331{Phos}gsAfsgAfaCfCfaguaGfcAfgCfuGfcsUfsu 4520 VII of sense strand D-3664Formula 3′ end {Phos}GfscsAfgCfuGfcUfAfCfUfgGfuUfcUfcUfu 4332{Phos}GfsasGfaAfcCfaguaGfcAfgCfuGfcsUfsu 4521 VII of sense strand D-3665Formula 3′ end {Phos}GfscsAfgCfuGfCfUfAfdCuGfgUfuCfuCfuUf 4333{Phos}gsAfsgAfaCfcAfguagcAfgCfuGfcsUfsu 4522 VII of sense strand D-3666Formula 3′ end {Phos}GfscsAfgCfuGfCfdTAfCfuGfgUfuCfuCfuUf 4334{Phos}gsAfsgAfaCfcAfguagcAfgCfuGfcsUfsu 4522 VII of sense strand D-3667Formula 3′ end {Phos}GfscsAfgCfuGfcdTAfCfUfGfgUfuCfuCfuUf 4335{Phos}gsAfsgAfaCfcaguaGfcAfgCfuGfcsUfsu 4523 VII of sense strand D-3668Formula 3′ end {Phos}GfscsAfgCfuGfcUfAfdCUfGfgUfuCfuCfuUf 4336{Phos}gsAfsgAfaCfcaguaGfcAfgCfuGfcsUfsu 4523 VII of sense strand D-3669Formula 3′ end {Phos}GfscsAfgCfugCfUfadCuGfgUfuCfuCfuUf 4337{Phos}gsAfsgAfaCfcAfgUfagCfAfgCfuGfcsUfsu 4524 VII of sense strandD-3670 Formula 3′ end {Phos}GfscsAfgCfugCfUfdACfuGfgUfuCfuCfuUf 4338{Phos}gsAfsgAfaCfcAfgUfagCfAfgCfuGfcsUfsu 4524 VII of sense strandD-3671 Formula 3′ end {Phos}GfscsAfgCfugCfdTaCfuGfgUfuCfuCfuUf 4339{Phos}gsAfsgAfaCfcAfgUfagCfAfgCfuGfcsUfsu 4524 VII of sense strandD-3672 Formula 3′ end {Phos}gscsagcuGfcUfadCugguucucuu 4340{Phos}gsAfsgAfaCfcAfgUfaGfcAfgCfugcsusu 4525 VII of sense strand D-3673Formula 3′ end {Phos}GfscsAfgCfuGfCfuAfCfuGfguucuCfuu 4341{Phos}gsAfsgAfaCfcAfguagcAfgCfuGfcsUfsu 4522 VII of sense strand D-3674Formula 3′ end {Phos}UfsgsUfgGfgAfaGfaAfaGfaUfgAfaGfuUf 4342{Phos}csUfsuCfaUfcUfuUfcUfuCfcCfaCfasUfsu 4526 VII of sense strandD-3675 Formula 3′ end {Phos}UfsgsUfgGfgaAfGfAfAfaGfaUfgAfaGfuUf 4343{Phos}csUfsuCfaUfcUfuucuUfCfcCfaCfasUfsu 4527 VII of sense strand D-3676Formula 3′ end {Phos}usgsugggAfaGfadAagaugaaguu 4344{Phos}csUfsuCfaUfcUfuUfcUfuCfcCfacasusu 4528 VII of sense strand D-3677Formula 3′ end {Phos}usgsugGfgAfAfGfAfAfaGfaugaaGfuu 4345{Phos}csUfsuCfaUfcUfuucuuCfcCfaCfasUfsu 4529 VII of sense strand D-3678Formula 3′ end {Phos}GfuGfgGfaAfGfAfAfAfgAfuGfaAfgUfuUf 4346{Phos}aCfuUfcAfuCfuuucuUfcCfcAfcUfu 4530 VII of sense strand D-3679Formula 3′ end {Phos}GfcAfgCfuGfCfUfAfCfuGfgUfuCfuCfuUf 4347{Phos}gAfgAfaCfcAfguagcAfgCfuGfcUfu 4531 VII of sense strand D-3680Formula 3′ end {Phos}UfgUfgGfgAfAfGfAfAfaGfaUfgAfaGfuUf 4348{Phos}cUfuCfaUfcUfuucuuCfcCfaCfaUfu 4532 VII of sense strand D-3681Formula 3′ end {Phos}AfgGfaCfuGfUfGfCfCfcAfcUfuCfaCfuUf 4349{Phos}gUfgAfaGfuGfggcacAfgUfcCfuUfu 4533 VII of sense strand D-3682Formula 3′ end {Phos}GfaGfaCfgGfGfCfUfUfcAfaGfaAfcUfuUf 4350{Phos}aGfuUfcUfuGfaagccCfgUfcUfcUfu 4534 VII of sense strand D-3683Formula 3′ end {Phos}GfaGfcGfcAfGfCfUfGfcUfaCfuGfgUfuUf 4351{Phos}aCfcAfgUfaGfcagcuGfcGfcUfcUfu 4535 VII of sense strand D-3684Formula 3′ end {Phos}GfuUfgUfcUfGfUfGfUfgAfuCfgGfaUfuUf 4352{Phos}aUfcCfgAfuCfacacaGfaCfaAfcUfu 4536 VII of sense strand D-3685Formula 3′ end {Phos}GfgAfgCfuGfCfGfGfGfgCfcUfgAfgAfuUf 4353{Phos}uCfuCfaGfgCfcccgcAfgCfuCfcUfu 4537 VII of sense strand D-3686Formula 3′ end {Phos}GfcCfgCfuGfGfAfAfCfgAfcGfaCfgUfuUf 4354{Phos}aCfgUfcGfuCfguuccAfgCfgGfcUfu 4538 VII of sense strand D-3687Formula 3′ end {Phos}GfcUfgGfgUfCfUfGfCfgAfgAfcAfgAfuUf 4355{Phos}uCfuGfuCfuCfgcagaCfcCfaGfcUfu 4539 VII of sense strand D-3688Formula 3′ end {Phos}GfcUfuUfcUfCfGfGfGfaAfuUfuUfcAfuUf 4356{Phos}uGfaAfaAfuUfcccgaGfaAfaGfcUfu 4540 VII of sense strand D-3689Formula 3′ end {Phos}CfcUfcCfuGfCfUfGfCfuUfgUfgGfuUfuUf 4357{Phos}aAfcCfaCfaAfgcagcAfgGfaGfgUfu 4541 VII of sense strand D-3690Formula 3′ end {Phos}CfuAfuCfaUfGfAfCfCfaAfgGfaGfuAfuUf 4358{Phos}uAfcUfcCfuUfggucaUfgAfuAfgUfu 4542 VII of sense strand D-3691Formula 3′ end {Phos}CfgUfcCfuGfGfGfAfGfgAfgCfaGfaAfuUf 4359{Phos}uUfcUfgCfuCfcucccAfgGfaCfgUfu 4543 VII of sense strand D-3692Formula 3′ end {Phos}CfuGfgGfgGfCfCfUfCfuUfcUfgCfuUfuUf 4360{Phos}aAfgCfaGfaAfgaggcCfcCfcAfgUfu 4544 VII of sense strand D-3693Formula 3′ end {Phos}CfcUfaUfcAfUfGfAfCfcAfaGfgAfgUfuUf 4361{Phos}aCfuCfcUfuGfgucauGfaUfaGfgUfu 4545 VII of sense strand D-3694Formula 3′ end {Phos}GfaUfaGfgGfUfGfAfUfgUfuCfcGfaAfuUf 4362{Phos}uUfcGfgAfaCfaucacCfcUfaUfcUfu 4546 VII of sense strand D-3695Formula 3′ end {Phos}GfcAfgUfuUfGfCfAfGfgUfuAfuCfaUfuUf 4363{Phos}aUfgAfuAfaCfcugcaAfaCfuGfcUfu 4547 VII of sense strand D-3696Formula 3′ end {Phos}GfsusGfgGfaAfgAfAfAfgAfuGfaAfgUfcGf 4364{Phos}csGfsaCfuUfcAfuCfuuuCfuUfcCfcAfcsUfsu 4548 VII of sense strandD-3697 Formula 3′ end {Phos}GfscsAfgCfuGfcUfAfCfuGfgUfuCfuCfuCf 4365{Phos}gsAfsgAfgAfaCfcAfguaGfcAfgCfuGfcsUfsu 4549 VII of sense strandD-3698 Formula 3′ end {Phos}UfsgsUfgGfgAfaGfAfAfaGfaUfgAfaGfuCf 4366{Phos}gsAfscUfuCfaUfcUfuucUfuCfcCfaCfasUfsu 4550 VII of sense strandD-3699 Formula 3′ end {Phos}AfsusGfuGfgGfaAfGfAfaAfgAfuGfaAfgUf 4367{Phos}asCfsuUfcAfuCfuUfucuUfcCfcAfcAfusUfsu 4551 VII of sense strandD-3700 Formula 3′ end {Phos}GfscsGfcAfgCfuGfCfUfaCfuGfgUfuCfuCf 4368{Phos}gsAfsgAfaCfcAfgUfagcAfgCfuGfcGfcsUfsu 4552 VII of sense strandD-3701 Formula 3′ end {Phos}AfsasUfgUfgGfgAfAfGfaAfaGfaUfgAfaGf 4369{Phos}csUfsuCfaUfcUfuUfcuuCfcCfaCfaUfusUfsu 4553 VII of sense strandD-3702 Formula 3′ end {Phos}GfsasCfgGfgAfcGfGfAfCfUfaCfgAfgAfuUf 4370{Phos}usCfsuCfgUfaguccGfuCfcCfgUfcsUfsu 4554 VII of sense strand D-3703Formula 3′ end {Phos}AfsgsCfcAfcCfuCfUfCfCfUfuUfaAfuUfuUf 4371{Phos}asAfsuUfaAfaggagAfgGfuGfgCfusUfsu 4555 VII of sense strand D-3704Formula 3′ end {Phos}UfsgsAfcCfuGfcGfGfAfGfCfcUfgAfgCfuUf 4372{Phos}gsCfsuCfaGfgcuccGfcAfgGfuCfasUfsu 4556 VII of sense strand D-3705Formula 3′ end {Phos}CfscsUfgCfuCfuCfCfCfUfGfgGfcCfuCfuUf 4373{Phos}gsAfsgGfcCfcagggAfgAfgCfaGfgsUfsu 4557 VII of sense strand D-3706Formula 3′ end {Phos}CfscsUfcCfuGfcUfCfUfCfCfcUfgGfgCfuUf 4374{Phos}gsCfscCfaGfggagaGfcAfgGfaGfgsUfsu 4558 VII of sense strand D-3707Formula 3′ end {Phos}GfscsUfgCfuAfcUfGfGfUfUfcUfcUfcGfuUf 4375{Phos}csGfsaGfaGfaaccaGfuAfgCfaGfcsUfsu 4559 VII of sense strand D-3708Formula 3′ end {Phos}CfscsCfaUfuCfuCfCfAfAfGfcUfuCfaGfuUf 4376{Phos}csUfsgAfaGfcuuggAfgAfaUfgGfgsUfsu 4560 VII of sense strand D-3709Formula 3′ end {Phos}AfscsUfgGfuUfcUfCfUfCfGfcUfcCfgGfuUf 4377{Phos}csCfsgGfaGfcgagaGfaAfcCfaGfusUfsu 4561 VII of sense strand D-3710Formula 3′ end {Phos}GfsasCfgGfgAfCfGfGfAfcUfaCfgAfgAfuUf 4378{Phos}usCfsuCfgUfaGfuccguCfcCfgUfcsUfsu 4562 VII of sense strand D-3711Formula 3′ end {Phos}AfsgsCfcAfcCfUfCfUfCfcUfuUfaAfuUfuUf 4379{Phos}asAfsuUfaAfaGfgagagGfuGfgCfusUfsu 4563 VII of sense strand D-3712Formula 3′ end {Phos}GfscsAfcGfaGfCfGfCfAfgCfuGfcUfaCfuUf 4380{Phos}gsUfsaGfcAfgCfugcgcUfcGfuGfcsUfsu 4564 VII of sense strand D-3713Formula 3′ end {Phos}GfscsGfcAfgCfUfGfCfUfaCfuGfgUfuCfuUf 4381{Phos}gsAfsaCfcAfgUfagcagCfuGfcGfcsUfsu 4565 VII of sense strand D-3714Formula 3′ end {Phos}AfscsGfgGfaCfGfGfAfCfuAfcGfaGfaCfuUf 4382{Phos}gsUfscUfcGfuAfguccgUfcCfcGfusUfsu 4566 VII of sense strand D-3715Formula 3′ end {Phos}GfscsUfcCfaCfGfUfGfAfaGfcAfgUfuCfuUf 4383{Phos}gsAfsaCfuGfcUfucacgUfgGfaGfcsUfsu 4567 VII of sense strand D-3716Formula 3′ end {Phos}UfsgsAfcCfuGfCfGfGfAfgCfcUfgAfgCfuUf 4384{Phos}gsCfsuCfaGfgCfuccgcAfgGfuCfasUfsu 4568 VII of sense strand D-3717Formula 3′ end {Phos}GfscsUfgCfgGfGfGfCfCfuGfaGfaGfaGfuUf 4385{Phos}csUfscUfcUfcAfggcccCfgCfaGfcsUfsu 4569 VII of sense strand D-3718Formula 3′ end {Phos}UfsusCfaCfcGfAfCfGfAfcGfgCfcGfcUfuUf 4386{Phos}asGfscGfgCfcGfucgucGfgUfgAfasUfsu 4570 VII of sense strand D-3719Formula 3′ end {Phos}CfscsAfcGfaCfCfAfAfAfaCfgGfgCfcCfuUf 4387{Phos}gsGfsgCfcCfgUfuuuggUfcGfuGfgsUfsu 4571 VII of sense strand D-3720Formula 3′ end {Phos}CfscsUfgCfuCfUfCfCfCfuGfgGfcCfuCfuUf 4388{Phos}gsAfsgGfcCfcAfgggagAfgCfaGfgsUfsu 4572 VII of sense strand D-3721Formula 3′ end {Phos}CfscsUfcCfuGfCfUfCfUfcCfcUfgGfgCfuUf 4389{Phos}gsCfscCfaGfgGfagagcAfgGfaGfgsUfsu 4573 VII of sense strand D-3722Formula 3′ end {Phos}GfsgsGfaAfgAfAfAfGfAfuGfaAfgUfcGfuUf 4390{Phos}csGfsaCfuUfcAfucuuuCfuUfcCfcsUfsu 4574 VII of sense strand D-3723Formula 3′ end {Phos}CfscsAfcUfuCfAfCfCfGfaCfgAfcGfgCfuUf 4391{Phos}gsCfscGfuCfgUfcggugAfaGfuGfgsUfsu 4575 VII of sense strand D-3724Formula 3′ end {Phos}GfscsUfgCfuAfCfUfGfGfuUfcUfcUfcGfuUf 4392{Phos}csGfsaGfaGfaAfccaguAfgCfaGfcsUfsu 4576 VII of sense strand D-3725Formula 3′ end {Phos}AfsasCfgGfgCfCfCfUfGfgAfaGfuGfgGfuUf 4393{Phos}csCfscAfcUfuCfcagggCfcCfgUfusUfsu 4577 VII of sense strand D-3726Formula 3′ end {Phos}AfsgsCfaGfcCfGfGfAfCfgAfcUfgGfuAfuUf 4394{Phos}usAfscCfaGfuCfguccgGfcUfgCfusUfsu 4578 VII of sense strand D-3727Formula 3′ end {Phos}CfsusGfcGfaGfAfCfAfGfaGfcUfgGfaCfuUf 4395{Phos}gsUfscCfaGfcUfcugucUfcGfcAfgsUfsu 4579 VII of sense strand D-3728Formula 3′ end {Phos}GfsgsAfgGfaCfGfCfGfCfaCfcUfgGfuGfuUf 4396{Phos}csAfscCfaGfgUfgcgcgUfcCfuCfcsUfsu 4580 VII of sense strand D-3729Formula 3′ end {Phos}UfscsAfcGfuCfCfUfGfGfgAfgGfaGfcAfuUf 4397{Phos}usGfscUfcCfuCfccaggAfcGfuGfasUfsu 4581 VII of sense strand D-3730Formula 3′ end {Phos}AfsasGfgAfcCfUfGfCfUfgCfcCfgGfuCfuUf 4398{Phos}gsAfscCfgGfgCfagcagGfuCfcUfusUfsu 4582 VII of sense strand D-3731Formula 3′ end {Phos}CfscsCfaUfuCfUfCfCfAfaGfcUfuCfaGfuUf 4399{Phos}csUfsgAfaGfcUfuggagAfaUfgGfgsUfsu 4583 VII of sense strand D-3732Formula 3′ end {Phos}AfsusGfgGfcCfUfCfCfAfcGfaCfcAfaAfuUf 4400{Phos}usUfsuGfgUfcGfuggagGfcCfcAfusUfsu 4584 VII of sense strand D-3733Formula 3′ end {Phos}CfsgsAfcGfgCfCfGfCfUfgGfaAfcGfaCfuUf 4401{Phos}gsUfscGfuUfcCfagcggCfcGfuCfgsUfsu 4585 VII of sense strand D-3734Formula 3′ end {Phos}AfscsUfgGfuUfCfUfCfUfcGfcUfcCfgGfuUf 4402{Phos}csCfsgGfaGfcGfagagaAfcCfaGfusUfsu 4586 VII of sense strand D-3735Formula 3′ end {Phos}CfsgsAfcCfaAfAfAfCfGfgGfcCfcUfgGfuUf 4403{Phos}csCfsaGfgGfcCfcguuuUfgGfuCfgsUfsu 4587 VII of sense strand D-3736Formula 3′ end {Phos}GfsgsUfcUfgCfGfAfGfAfcAfgAfgCfuGfuUf 4404{Phos}csAfsgCfuCfuGfucucgCfaGfaCfcsUfsu 4588 VII of sense strand D-3737Formula 3′ end {Phos}GfaCfgGfgAfCfGfGfAfcUfaCfgAfgAfuUf 4405{Phos}uCfuCfgUfaGfuccguCfcCfgUfcUfu 4589 VII of sense strand D-3738Formula 3′ end {Phos}AfgCfcAfcCfUfCfUfCfcUfuUfaAfuUfuUf 4406{Phos}aAfuUfaAfaGfgagagGfuGfgCfuUfu 4590 VII of sense strand D-3739Formula 3′ end {Phos}UfgAfcCfuGfCfGfGfAfgCfcUfgAfgCfuUf 4407{Phos}gCfuCfaGfgCfuccgcAfgGfuCfaUfu 4591 VII of sense strand D-3740Formula 3′ end {Phos}CfcUfgCfuCfUfCfCfCfuGfgGfcCfuCfuUf 4408{Phos}gAfgGfcCfcAfgggagAfgCfaGfgUfu 4592 VII of sense strand D-3741Formula 3′ end {Phos}CfcUfcCfuGfCfUfCfUfcCfcUfgGfgCfuUf 4409{Phos}gCfcCfaGfgGfagagcAfgGfaGfgUfu 4593 VII of sense strand D-3742Formula 3′ end {Phos}GfcUfgCfuAfCfUfGfGfuUfcUfcUfcGfuUf 4410{Phos}cGfaGfaGfaAfccaguAfgCfaGfcUfu 4594 VII of sense strand D-3743Formula 3′ end {Phos}CfcCfaUfuCfUfCfCfAfaGfcUfuCfaGfuUf 4411{Phos}cUfgAfaGfcUfuggagAfaUfgGfgUfu 4595 VII of sense strand D-3744Formula 3′ end {Phos}AfcUfgGfuUfCfUfCfUfcGfcUfcCfgGfuUf 4412{Phos}cCfgGfaGfcGfagagaAfcCfaGfuUfu 4596 VII of sense strand D-3745Formula 5′ end GfcAfgCfuGfcUfAfCfUfggUfuCfuCfsusUf 4413{Phos}gsAfsgAfaCfCfaguaGfcAfgCfuGfcsUfsu 4520 XVI, k = 3, of n = 1 sensestrand D-3746 Formula 3′ end {Phos}GfsusGfgGfaAfgAfAfAfGfauGfaAfgUfuUf4414 {Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4597 VIII of sense strandD-3747 Formula 3′ end {Phos}GfsusGfgGfaAfgAfAfAfGfAfuGfaAfgUfuUf 4415{Phos}asCfsuUfcAfucuuuCfuUfcCfcAfcsUfsu 4514 VIII of sense strand D-3748Formula 5′ end GfuGfgGfaAfgAfAfAfGfAfuGfaAfgUfsusUf 4416{Phos}asCfsuUfcAfucuuuCfuUfcCfcAfcsUfsu 4514 XVI, k = 3, of n = 1 sensestrand D-3749 Formula 3′ end {Phos}GfuGfgGfaaGfAfaAfgAfuGfaAfgUfuUf 4417{Phos}aCfuUfcAfuCfuUfucUfUfcCfcAfcUfu 4598 VIII of sense strand D-3750Formula 5′ end GfuGfgGfaaGfAfaAfgAfuGfaAfgUfsusUf 4418{Phos}asCfsuUfcAfuCfuUfucUfUfcCfcAfcsUfsu 4515 XVI, k = 3, of n = 1sense strand D-3751 Formula 5′ end GfuGfgGfaAfgAfaAfGfauGfaAfgUfsusUf4419 {Phos}asCfsuUfcAfUfcuUfuCfuUfcCfcAfcsUfsu 4599 XVI, k = 3, of n = 1sense strand D-3752 Formula 5′ end GfuGfgGfaAfgAfAfAfgAfuGfaAfgUfsusUf4420 {Phos}asCfsuUfcAfuCfuuuCfuUfcCfcAfcsUfsu 4600 XXVI of sense strandD-3753 None N/A {Phos}GfuGfgGfaAfgAfAfAfGfAfuGfaAfgUfuUf 4421{Phos}aCfuUfcAfucuuuCfuUfcCfcAfcUfu 4601 D-3754 None N/A{Phos}GfuGfgGfaAfgAfaAfgAfuGfaAfguUfUf 4422{Phos}AfCfuUfcAfuCfuUfuCfuUfcCfcAfcUfu 4602 D-3755 None N/A{Phos}GfuGfgGfaAfgAfAfAfGfauGfaAfgUfuUf 4423{Phos}aCfuUfcAfUfcuuuCfuUfcCfcAfcUfu 4603 D-3756 None N/A{Phos}GfsusGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf 4424{Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4597 D-3757 None N/A{Phos}GfuGfgGfaAfgAfAfAfGfaUfgAfaGfuUfu 4425{Phos}AfcUfuCfaUfcuuuCfuUfcCfcAfcUfu 4604 D-3758 Formula 3′ end{Phos}GfscsAfgCfuGfcUfAfCfUfGfgUfuCfuCfuUf 4426{Phos}gsAfsgAfaCfcaguaGfcAfgCfuGfcsUfsu 4523 VIII of sense strand D-3759Formula 3′ end {Phos}UfsgsUfgGfgAfaGfAfAfAfGfaUfgAfaGfuUf 4427{Phos}csUfsuCfaUfcuuucUfuCfcCfaCfasUfsu 4605 VIII of sense strand D-3760Formula 3′ end {Phos}GfsusGfgGfaaGfAfaAfgAfuGfaAfgUfuUf 4428{Phos}asCfsuUfcAfuCfuUfucUfUfcCfcAfcsUfsu 4515 VIII of sense strandD-3761 Formula 3′ end {Phos}GfscsAfgCfugCfUfaCfuGfgUfuCfuCfuUf 4429{Phos}gsAfsgAfaCfcAfgUfagCfAfgCfuGfcsUfsu 4524 VIII of sense strandD-3762 Formula 3′ end {Phos}GfsusGfgGfaAfgAfaAfgAfuGfaAfgUfuUf 4430{Phos}asCfsuUfcAfuCfuUfuCfuUfcCfcAfcsUfsUf 4606 VIII of sense strandD-3763 Formula 3′ end {Phos}GfsusGfgGfaAfgAfaAfGfauGfaAfgUfuUf 4431{Phos}asCfsuUfcAfUfcuUfuCfuUfcCfcAfcsUfsu 4599 VIII of sense strandD-3764 Formula 3′ end {Phos}GfsusGfggAfAfgAfaAfgAfuGfaAfgUfuUf 4432{Phos}asCfsuUfcAfuCfuUfuCfuuCfCfcAfcsUfsu 4607 VIII of sense strandD-3765 Formula 3′ end {Phos}GfsusgGfGfaAfgAfaAfgAfuGfaAfgUfuUf 4433{Phos}asCfsuUfcAfuCfuUfuCfuUfccCfAfcsUfsu 4608 VIII of sense strandD-3766 Formula 3′ end {Phos}gsUfsGfgGfaAfgAfaAfgAfuGfaAfgUfuUf 4434{Phos}asCfsuUfcAfuCfuUfuCfuUfcCfcaCfsUfsUf 4609 VIII of sense strandD-3767 Formula 5′ end GfuGfgGfaAfgAfaAfgAfUfgaAfgUfsusUf 4435{Phos}asCfsuUfCfauCfuUfuCfuUfcCfcAfcsUfsu 4610 XVI, k = 3, of n = 1sense strand D-3768 Formula 5′ end GfuGfgGfaAfgAfaAfgAfuGfAfagUfsusUf4436 {Phos}asCfsUfucAfuCfuUfuCfuUfcCfcAfcsUfsu 4611 XVI, k = 3, of n = 1sense strand D-3769 Formula 5′ end GfuGfgGfaAfgAfaAfgAfuGfaAfGfususUf4437 {Phos}AfscsuUfcAfuCfuUfuCfuUfcCfcAfcsUfsu 4612 XVI, k = 3, of n = 1sense strand D-3770 Formula 5′ end gUfgGfgAfaGfAfAfAfgAfuGfaAfgUfsusUf4438 {Phos}asCfsuUfcAfuCfuuucUfuCfcCfaCfsusUf 4613 XVI, k = 3, of n = 1sense strand D-3771 Formula 5′ end GfuGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf4439 {Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4597 XVI, k = 3, of n = 1sense strand D-3772 Formula 5′ end GfuGfgGfaAfgAfAfAfGfaUfgAfaGfusUfsu4440 {Phos}AfscsUfuCfaUfcuuuCfuUfcCfcAfcsUfsu 4614 XVI, k = 3, of n = 1sense strand D-3773 Formula 5′ end GfcAfgCfuGfcUfAfCfUfGfgUfuCfuCfsusUf4441 {Phos}gsAfsgAfaCfcaguaGfcAfgCfuGfcsUfsu 4523 XVI, k = 3, of n = 1sense strand D-3774 Formula 3′ end{Phos}GfsusGfgGfaAfgAfAfAfGfauGfaAfgUfuUf 4414{Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4597 XXIX of sense strandD-3775 Formula 3′ end {Phos}GfsusGfgGfaAfgAfaAfGfauGfaAfgUfuUf 4431{Phos}asCfsuUfcAfUfcuUfuCfuUfcCfcAfcsUfsu 4599 XXIX of sense strandD-3776 None N/A {Phos}GfscsAfgCfugCfUfaCfuGfgUfuCfuCfuUf 4429{Phos}gsAfsgAfaCfcAfgUfagCfAfgCfuGfcsUfsUf 4615 D-3777 None N/A{Phos}GfscsAfgCfuGfcUfAfCfUfGfgUfuCfuCfuUf 4426{Phos}gsAfsgAfaCfcaguaGfcAfgCfuGfcsUfsUf 4616 D-3778 Formula 3′ end{Phos}GfscsAfgCfuGfcUfaCfugGfUfuCfuCfuUf 4442{Phos}gsAfsgAfacCfAfgUfaGfcAfgCfuGfcsUfsu 4617 VIII of sense strandD-3779 Formula 5′ end GfcCfgCfuGfgAfAfCfGfacGfaCfgUfsusUf 4443{Phos}asCfsgUfcGfUfcguuCfcAfgCfgGfcsUfsu 4618 XVI, k = 3, of n = 1 sensestrand D-3780 Formula 5′ end GfaUfaGfgGfuGfAfUfGfuuCfcGfaAfsusUf 4444{Phos}usUfscGfgAfAfcaucAfcCfcUfaUfcsUfsu 4619 XVI, k = 3, of n = 1 sensestrand D-3781 Formula 5′ end CfcUfaUfcAfuGfAfCfCfaaGfgAfgUfsusUf 4445{Phos}asCfsuCfcUfUfggucAfuGfaUfaGfgsUfsu 4620 XVI, k = 3, of n = 1 sensestrand D-3782 Formula 5′ end CfuAfuCfaUfgAfCfCfAfagGfaGfuAfsusUf 4446{Phos}usAfscUfcCfUfugguCfaUfgAfuAfgsUfsu 4621 XVI, k = 3, of n = 1 sensestrand D-3783 Formula 5′ end AfaCfgGfgCfcCfUfGfGfaaGfuGfgGfsusUf 4447{Phos}csCfscAfcUfUfccagGfgCfcCfgUfusUfsu 4622 XVI, k = 3, of n = 1 sensestrand D-3784 Formula 5′ end GfaCfgGfgAfcGfGfAfCfuaCfgAfgAfsusUf 4448{Phos}usCfsuCfgUfAfguccGfuCfcCfgUfcsUfsu 4623 XVI, k = 3, of n = 1 sensestrand D-3785 Formula 5′ end AfcGfgGfaCfgGfAfCfUfacGfaGfaCfsusUf 4449{Phos}gsUfscUfcGfUfagucCfgUfcCfcGfusUfsu 4624 XVI, k = 3, of n = 1 sensestrand D-3786 Formula 5′ end AfgCfaGfcCfgGfAfCfGfacUfgGfuAfsusUf 4450{Phos}usAfscCfaGfUfcgucCfgGfcUfgCfusUfsu 4625 XVI, k = 3, of n = 1 sensestrand D-3787 Formula 5′ end CfcAfcUfuCfaCfCfGfAfcgAfcGfgCfsusUf 4451{Phos}gsCfscGfuCfGfucggUfgAfaGfuGfgsUfsu 4626 XVI, k = 3, of n = 1 sensestrand D-3788 Formula 5′ end CfgAfcGfgCfcGfCfUfGfgaAfcGfaCfsusUf 4452{Phos}gsUfscGfuUfCfcagcGfgCfcGfuCfgsUfsu 4627 XVI, k = 3, of n = 1 sensestrand D-3789 Formula 5′ end AfaAfcCfcGfuGfGfCfGfcuUfuCfuGfsusUf 4453{Phos}csAfsgAfaAfGfcgccAfcGfgGfuUfusUfsu 4628 XVI, k = 3, of n = 1 sensestrand D-3790 Formula 5′ end CfcUfcUfuCfuGfCfUfUfucUfcGfgGfsusUf 4454{Phos}csCfscGfaGfAfaagcAfgAfaGfaGfgsUfsu 4629 XVI, k = 3, of n = 1 sensestrand D-3791 Formula 5′ end GfaAfaCfcCfgUfGfGfCfgcUfuUfcUfsusUf 4455{Phos}asGfsaAfaGfCfgccaCfgGfgUfuUfcsUfsu 4630 XVI, k = 3, of n = 1 sensestrand D-3792 Formula 5′ end GfcAfgAfaAfuUfUfGfUfccAfgCfaCfsusUf 4456{Phos}gsUfsgCfuGfGfacaaAfuUfuCfuGfcsUfsu 4631 XVI, k = 3, of n = 1 sensestrand D-3793 Formula 5′ end GfgAfcUfaCfgAfGfAfCfggGfcUfuCfsusUf 4457{Phos}gsAfsaGfcCfCfgucuCfgUfaGfuCfcsUfsu 4632 XVI, k = 3, of n = 1 sensestrand D-3794 Formula 5′ end GfuGfcCfcAfcUfUfCfAfccGfaCfgAfsusUf 4458{Phos}usCfsgUfcGfGfugaaGfuGfgGfcAfcsUfsu 4633 XVI, k = 3, of n = 1 sensestrand D-3795 Formula 5′ end GfuUfgUfcUfgUfGfUfGfauCfgGfaUfsusUf 4459{Phos}asUfscCfgAfUfcacaCfaGfaCfaAfcsUfsu 4634 XVI, k = 3, of n = 1 sensestrand D-3796 Formula 5′ end GfgUfcUfgCfgAfGfAfCfagAfgCfuGfsusUf 4460{Phos}csAfsgCfuCfUfgucuCfgCfaGfaCfcsUfsu 4635 XVI, k = 3, of n = 1 sensestrand D-3797 Formula 5′ end AfgCfcAfcCfuCfUfCfCfuuUfaAfuUfsusUf 4461{Phos}asAfsuUfaAfAfggagAfgGfuGfgCfusUfsu 4636 XVI, k = 3, of n = 1 sensestrand D-3798 Formula 5′ end GfcUfcCfaCfgUfGfAfAfgcAfgUfuCfsusUf 4462{Phos}gsAfsaCfuGfCfuucaCfgUfgGfaGfcsUfsu 4637 XVI, k = 3, of n = 1 sensestrand D-3799 Formula 5′ end GfcGfcAfgCfuGfCfUfAfcuGfgUfuCfsusUf 4463{Phos}gsAfsaCfcAfGfuagcAfgCfuGfcGfcsUfsu 4638 XVI, k = 3, of n = 1 sensestrand D-3800 Formula 5′ end GfcUfgCfuAfcUfGfGfUfucUfcUfcGfsusUf 4464{Phos}csGfsaGfaGfAfaccaGfuAfgCfaGfcsUfsu 4639 XVI, k = 3, of n = 1 sensestrand D-3801 Formula 5′ end AfcUfgGfuUfcUfCfUfCfgcUfcCfgGfsusUf 4465{Phos}csCfsgGfaGfCfgagaGfaAfcCfaGfusUfsu 4640 XVI, k = 3, of n = 1 sensestrand D-3802 Formula 3′ end {Phos}GfcAfgCfuGfcUfAfCfUfGfgUfuCfuCfuUf4466 {Phos}gAfgAfaCfcaguaGfcAfgCfuGfcUfu 4641 VIII of sense strandD-3803 Formula 3′ end GfscsAfgCfuGfcUfAfCfUfGfgUfuCfuCfuUf 4467gsAfsgAfaCfcaguaGfcAfgCfuGfcsUfsu 4642 VIII of sense strand D-3804Formula 5′ end GfcAfGCfUGfcUfAfCfUfGfGUfUCfUCfUUf 4468{Phos}GAfGAfACfcAGUAGfcAfGCfUGfcUfu 4643 XVI, k = 3, of n = 1 sensestrand D-3805 Formula 3′ end {Phos}gsUfsgGfgAfaGfAfaAfgAfuGfaAfgUfuUf4469 {Phos}asCfsuUfcAfuCfuUfucUfuCfcCfaCfsusUf 4644 VIII of sense strandD-3806 Formula 5′ end gUfgGfgAfaGfAfaAfgAfuGfaAfgUfsusUf 4470{Phos}asCfsuUfcAfuCfuUfucUfuCfcCfaCfsusUf 4644 XVI, k = 3, of n = 1sense strand D-3807 Formula 5′ end gUfgGfgAfagAfaAfgAfuGfaAfgUfsusUf4471 {Phos}asCfsuUfcAfuCfuUfuCfUfuCfcCfaCfsusUf 4645 XVI, k = 3, ofn = 1 sense strand D-3808 Formula 5′ endAfuGfgGfcCfuCfCfAfCfgaCfcAfaAfsusUf 4472{Phos}usUfsuGfgUfCfguggAfgGfcCfcAfusUfsu 4646 XVI, k = 3, of n = 1 sensestrand D-3809 Formula 5′ end GfcAfcGfaGfcGfCfAfGfcuGfcUfaCfsusUf 4473{Phos}gsUfsaGfcAfGfcugcGfcUfcGfuGfcsUfsu 4647 XVI, k = 3, of n = 1 sensestrand D-3810 Formula 5′ end UfuCfaCfcGfaCfGfAfCfggCfcGfcUfsusUf 4474{Phos}asGfscGfgCfCfgucgUfcGfgUfgAfasUfsu 4648 XVI, k = 3, of n = 1 sensestrand D-3811 Formula 5′ end GfaAfgUfgGfgUfGfGfAfcgGfgAfcGfsusUf 4475{Phos}csGfsuCfcCfGfuccaCfcCfaCfuUfcsUfsu 4649 XVI, k = 3, of n = 1 sensestrand D-3812 Formula 5′ end UfcAfcGfuCfcUfGfGfGfagGfaGfcAfsusUf 4476{Phos}usGfscUfcCfUfcccaGfgAfcGfuGfasUfsu 4650 XVI, k = 3, of n = 1 sensestrand D-3813 Formula 5′ end GfuGfgGfaAfgAfAfAfGfauGfaAfgUfuUfs{invAb}4477 {Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4597 XVI, k = 3, of n = 1sense strand D-3814 Formula 5′ end[invAb]GfuGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf 4478{Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4597 XVI, k = 3, of n = 1 sensestrand D-3815 Formula 5′ end [invAb]GfuGfgGfaAfgAfAfAfGfauGfaAfgUfuUfs4479 {Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4597 XVI, k = 3, of{invAb} n = 1 sense strand D-3816 Formula 5′ endGfuGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf 4439{Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcUfus 4651 XVI, k = 3, of {invAb}n = 1 sense strand D-3817 Formula 5′ endGfsusGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf 4480{Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4597 XVI, k = 3, of n = 1 sensestrand D-3818 Formula 5′ end GfsusGfsgGfaAfgAfAfAfGfauGfaAfgUfsusUf 4481{Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4597 XVI, k = 3, of n = 1 sensestrand D-3819 Formula 5′ end GfuGfgGfaAfgAfAfAfGfauGfaAfgsUfsusUf 4482{Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4597 XVI, k = 3, of n = 1 sensestrand D-3820 Formula 5′ end GfuGfgGfaAfgAfAfAfGfauGfaAfgUfusUf 4483{Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4597 XVI, k = 3, of n = 1 sensestrand D-3821 Formula 5′ end GfuGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf 4439{Phos}asCfsusUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4652 XVI, k = 3, of n = 1sense strand D-3822 Formula 5′ end GfuGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf4439 {Phos}asCfuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4653 XVI, k = 3, of n = 1sense strand D-3823 Formula 5′ end GfuGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf4439 {Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfscsUfsu 4654 XVI, k = 3, of n = 1sense strand D-3824 Formula 5′ end GfuGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf4439 {Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcUfsu 4655 XVI, k = 3, of n = 1sense strand D-3825 Formula 5′ end GfuGfgGfaAfgAfAfAfGfauGfaAfgUfuUf4484 {Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4597 XVI, k = 3, of n = 1sense strand D-3826 Formula 5′ end GfuGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf4439 {Phos}aCfuUfcAfUfcuuuCfuUfcCfcAfcsUfsu 4656 XVI, k = 3, of n = 1sense strand D-3827 Formula 5′ end GfuGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf4439 {Phos}asCfsuUfcAfUfcuuuCfuUfcCfcAfcUfu 4657 XVI, k = 3, of n = 1sense strand D-3828 Formula 5′ end CfcGfcUfgGfaAfCfGfAfcgAfcGfuCfsusUf4485 {Phos}gsAfscGfuCfGfucguUfcCfaGfcGfgsUfsu 4658 XVI, k = 3, of n = 1sense strand D-3829 Formula 5′ end GfaUfgCfcAfcGfUfUfUfggCfgUfgCfsusUf4486 {Phos}gsCfsaCfgCfCfaaacGfuGfgCfaUfcsUfsu 4659 XVI, k = 3, of n = 1sense strand D-3830 Formula 5′ end GfaGfcAfcCfcAfGfGfGfagGfcAfaUfsusUf4487 {Phos}asUfsuGfcCfUfcccuGfgGfuGfcUfcsUfsu 4660 XVI, k = 3, of n = 1sense strand D-3831 Formula 5′ end GfgCfuUfgAfgCfAfCfCfcaGfgGfaGfsusUf4488 {Phos}csUfscCfcUfGfggugCfuCfaAfgCfcsUfsu 4661 XVI, k = 3, of n = 1sense strand D-3832 Formula 5′ end GfaAfgAfaAfgAfUfGfAfagUfcGfcUfsusUf4489 {Phos}asGfscGfaCfUfucauCfuUfuCfuUfcsUfsu 4662 XVI, k = 3, of n = 1sense strand D-3833 Formula 5′ end GfaGfaUfgCfcAfCfGfUfuuGfgCfgUfsusUf4490 {Phos}asCfsgCfcAfAfacguGfgCfaUfcUfcsUfsu 4663 XVI, k = 3, of n = 1sense strand D-3834 Formula 5′ end CfgGfgAfcGfgAfCfUfAfcgAfgAfcGfsusUf4491 {Phos}csGfsuCfuCfGfuaguCfcGfuCfcCfgsUfsu 4664 XVI, k = 3, of n = 1sense strand D-3835 Formula 5′ end CfuAfcGfaGfaCfGfGfGfcuUfcAfaGfsusUf4492 {Phos}csUfsuGfaAfGfcccgUfcUfcGfuAfgsUfsu 4665 XVI, k = 3, of n = 1sense strand D-3836 Formula 5′ end CfcCfgGfgCfaCfUfGfGfagAfuGfcCfsusUf4493 {Phos}gsGfscAfuCfUfccagUfgCfcCfgGfgsUfsu 4666 XVI, k = 3, of n = 1sense strand D-3837 Formula 5′ end UfgAfaAfcCfcGfUfGfGfcgCfuUfuCfsusUf4494 {Phos}gsAfsaAfgCfGfccacGfgGfuUfuCfasUfsu 4667 XVI, k = 3, of n = 1sense strand D-3838 Formula 5′ end GfgUfgGfuCfaCfGfUfCfcuGfgGfaGfsusUf4495 {Phos}csUfscCfcAfGfgcsgUfgAfcCfaCfcsUfsu 4668 XVI, k = 3, of n = 1sense strand D-3839 Formula 5′ end CfaGfaAfaUfuUfGfUfCfcaGfcAfcCfsusUf4496 {Phos}gsGfsuGfcUfGfgacaAfaUfuUfcUfgsUfsu 4669 XVI, k = 3, of n = 1sense strand D-3840 Formula 5′ end AfuGfcCfaCfgUfUfUfGfgcGfuGfcUfsusUf4497 {Phos}asGfscAfcGfCfcaaaCfgUfgGfcAfusUfsu 4670 XVI, k = 3, of n = 1sense strand D-3841 Formula 5′ end AfgCfuGfcGfgGfGfCfCfugAfgAfgAfsusUf4498 {Phos}usCfsuCfuCfAfggccCfcGfcAfgCfusUfsu 4671 XVI, k = 3, of n = 1sense strand D-3842 Formula 5′ end GfcCfcUfaUfcAfUfGfAfccAfaGfgAfsusUf4499 {Phos}usCfscUfuGfGfucauGfaUfaGfgGfcsUfsu 4672 XVI, k = 3, of n = 1sense strand D-3843 Formula 5′ end AfgCfaAfcUfuCfAfCfAfgcGfaGfcAfsusUf4500 {Phos}usGfscUfcGfCfugugAfaGfuUfgCfusUfsu 4673 XVI, k = 3, of n = 1sense strand D-3844 Formula 5′ end AfgCfaGfaAfaUfUfUfGfucCfaGfcAfsusUf4501 {Phos}usGfscUfgGfAfcaaaUfuUfcUfgCfusUfsu 4674 XVI, k = 3, of n = 1sense strand D-3845 Formula 5′ end AfaAfaCfgGfgCfCfCfUfggAfaGfuGfsusUf4502 {Phos}csAfscUfuCfCfagggCfcCfgUfuUfusUfsu 4675 XVI, k = 3, of n = 1sense strand D-3846 Formula 5′ end GfcCfuGfaGfcUfGfUfCfagAfuGfgCfsusUf4503 {Phos}gsCfscAfuCfUfgacaGfcUfcAfgGfcsUfsu 4676 XVI, k = 3, of n = 1sense strand D-3847 Formula 5′ end UfgGfaGfaUfgCfCfAfCfguUfuGfgCfsusUf4504 {Phos}gsCfscAfaAfCfguggCfaUfcUfcCfasUfsu 4677 XVI, k = 3, of n = 1sense strand D-3848 Formula 5′ end AfcUfaCfgAfgAfCfGfGfgcUfuCfaAfsusUf4505 {Phos}usUfsgAfaGfCfccguCfuCfgUfaGfusUfsu 4678 XVI, k = 3, of n = 1sense strand D-3849 Formula 5′ end AfaGfuGfgGfuGfGfAfCfggGfaCfgGfsusUf4506 {Phos}csCfsgUfcCfCfguccAfcCfcAfcUfusUfsu 4679 XVI, k = 3, of n = 1sense strand D-3850 Formula 5′ end UfgCfaCfaGfcAfCfUfGfaaGfaAfcCfsusUf4507 {Phos}gsGfsuUfcUfUfcaguGfcUfgUfgCfasUfsu 4680 XVI, k = 3, of n = 1sense strand D-3851 Formula 5′ end AfcGfgAfcUfaCfGfAfGfacGfgGfcUfsusUf4508 {Phos}asGfscCfcGfUfcucgUfaGfuCfcGfusUfsu 4681 XVI, k = 3, of n = 1sense strand D-3852 Formula 5′ end GfgAfcGfaCfuGfGfUfAfcgGfcCfaCfsusUf4509 {Phos}gsUfsgGfcCfGfuaccAfgUfcGfuCfcsUfsu 4682 XVI, k = 3, of n = 1sense strand D-3853 Formula 5′ end GfaGfcUfgCfgGfGfGfCfcuGfaGfaGfsusUf4510 {Phos}csUfscUfcAfGfgcccCfgCfaGfcUfcsUfsu 4683 XVI, k = 3, of n = 1sense strand D-3854 Formula 5′ end CfaCfgUfuUfgGfCfGfUfgcUfuGfgAfsusUf4511 {Phos}usCfscAfaGfCfacgcCfaAfaCfgUfgsUfsu 4684 XVI, k = 3, of n = 1sense strand D-3855 Formula 5′ end GfuGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf4439 {Phos}[invAbs]asCfsuUfcAfUfcuuuCfuUfcCfcAf 4685 XVI, k = 3, ofcsUfsu n = 1 sense strand D-3856 Formula 5′ endGfuGfgGfaAfgAfAfAfGfauGfaAfgUfsusUf 4439{Phos}[invAbs]asCfsuUfcAfUfcuuuCfuUfcCfcAf 4686 XVI, k = 3, ofcUfus{invAb} n = 1 sense strand D-3857 Formula 5′ endGfuGfgGfaaGfAfAfAfgAfuGfaAfgUfsusUf 4512{Phos}asCfsuUfcAfuCfuuucUfUfcCfcAfcsUfsu 4687 XVI, k = 3, of n = 1 sensestrand

The efficacy of the GalNAc-siRNA conjugates for inhibiting ASGR1expression was tested in the Hep3B cell transfection immunoassay(described in Example 2) and/or the human ASGR1 CHO cell free uptakeimmunoassay (described in Example 7). The results of the assays areshown in Table 9 below.

TABLE 9 Efficacy of GalNAc-ASGR1 siRNA conjugates in vitro Target siteof Target site of Hep3B hASGR1 hASGR1 CHO Free antisense antisense Hep3BTransfected Max CHO Free Uptake Max sequence within sequence withinDuplex Transfected Antagonist Uptake IC50 Antagonist NM_ 001671.4 NM_1197216.2 No. IC50 IP (μM) Activity IP (μM) Activity  692-710  575-593D-3651 0.00141 −0.90 >0.5 −0.51  692-710  575-593 D-3652 0.00239−0.97 >0.5 −0.34  692-710  575-593 D-3653 0.00115 −0.84 >0.5 −0.42 692-710  575-593 D-3654 0.00206 −0.89 >0.5 −0.32  692-710  575-593D-3655 0.00461 −0.77 >0.5 −0.15  692-710  575-593 D-3656 0.00476−0.97 >0.5 −0.13  692-710  575-593 D-3657 0.00431 −0.83 >0.5 −0.10 692-710  575-593 D-3658 0.00231 −0.96 >0.5 −0.35  692-710  575-593D-3659 0.00177 −0.90 >0.5 −0.48  888-906  771-789 D-3660 0.00398−0.62 >0.5 −0.04  888-906  771-789 D-3661 >12.5 −0.4 >25.0 −0.30 888-906  771-789 D-3662 >12.5 0.0 >25.0 −0.10  888-906  771-789 D-36630.00309 −0.9 >25.0 −0.20  888-906  771-789 D-3664 0.00229 −0.7 >25.00.10  888-906  771-789 D-3665 0.000862 −0.7 >25.0 0.00  888-906  771-789D-3666 0.000545 −0.8 >25.0 0.10  888-906  771-789 D-3667 0.000716−0.8 >25.0 0.10  888-906  771-789 D-3668 0.00358 −0.8 >25.0 0.10 888-906  771-789 D-3669 >12.5 −0.5 >25.0 −0.10  888-906  771-789D-3670 >12.5 −0.3 >25.0 0.10  888-906  771-789 D-3671 >12.5 −0.1 >25.0−0.10  888-906  771-789 D-3672 0.00156 −0.7 >25.0 0.10  888-906  771-789D-3673 >12.5 −0.2 >25.0 −0.20  691-709  574-592 D-3674 0.59834−0.9 >25.0 −0.10  691-709  574-592 D-3675 1 −0.5 >25.0 −0.10  691-709 574-592 D-3676 0.24304 −0.7 >25.0 −0.10  691-709  574-592 D-3677 1.99−0.9 >25.0 −0.30  692-710  575-593 D-3678 0.0085575 −1.0 1.26 −0.40 888-906  771-789 D-3679 0.33276 −0.8 0.00324 −0.20  691-709  574-592D-3680 0.079157 −1.0 >12.5 0.00 1158-1176 1041-1059 D-3681 0.02749−0.8 >12.5 −0.20 1088-1106  971-989 D-3682 0.0064167 −1.0 9.4 −0.30 884-902  767-785 D-3683 0.0057367 −0.9 >12.5 −0.30  563-581 — D-36840.18997 −1.0 5.48 −0.20  604-622  487-505 D-3685 0.070325 −0.9 0.306−0.10 1185-1203 1068-1086 D-3686 0.25317 −0.9 >12.5 −0.10 1221-12391104-1122 D-3687 0.012227 −0.9 >12.5 0.10 1349-1367 1232-1250 D-36880.002845 −1.0 >12.5 −0.20  547-565 — D-3689 0.009105 −0.8 >12.5 −0.20 396-414  396-414 D-3690 0.003015 −1.0 >12.5 −0.10  978-996  861-879D-3691 0.24201 −0.9 >12.5 0.10 1334-1352 1217-1235 D-3692 0.10186−0.9 >12.5 −0.10  395-413  395-413 D-3693 0.081347 −0.9 >12.5 −0.101393-1411 1276-1294 D-3694 0.58738 −1.0 >12.5 0.10 1443-1461 1326-1344D-3695 0.015662 −0.9 >12.5 0.20  692-710  575-593 D-3696 >12.5 0.1 >25.0−0.30  888-906  771-789 D-3697 0.00389 −0.7 >25.0 0.00  691-709  574-592D-3698 >12.5 −0.2 >25.0 −0.10  692-710  575-593 D-3699 0.00324 −0.90.638 −0.40  888-906  771-789 D-3700 >12.5 −0.6 >25.0 0.10  691-709 574-592 D-3701 >12.5 −0.5 >25.0 0.10 1073-1091  956-974 D-3702 0.009365−0.8 >12.5 −0.30 1260-1278 1143-1161 D-3703 >0.195 −0.4 >12.5 −0.20 796-814  679-697 D-3704 >12.5 −0.3 >12.5 −0.20  526-544 — D-3705 >12.5−0.2 >12.5 −0.10  523-541 — D-3706 >12.5 −0.2 >12.5 −0.20  891-909 774-792 D-3707 0.00932 −0.6 >12.5 −0.20  288-306  288-306 D-3708 2.22−0.5 >12.5 −0.20  897-915  780-798 D-3709 0.0129 −0.8 >12.5 −0.201073-1091  956-974 D-3710 0.00656 −0.9 >12.5 −0.20 1260-1278 1143-1161D-3711 >12.5 −0.2 >12.5 −0.10  880-898  763-781 D-3712 0.00684−0.6 >12.5 −0.20  886-904  769-787 D-3713 0.0102 −0.7 >12.5 −0.201074-1092  957-975 D-3714 0.005445 −0.8 >12.5 −0.30  772-790  655-673D-3715 >12.5 −0.2 >12.5 −0.20  796-814  679-697 D-3716 >12.5 −0.3 >12.5−0.40  607-625  490-508 D-3717 >12.5 −0.3 >12.5 −0.20 1172-11901055-1073 D-3718 >12.5 0.0 >12.5 −0.20 1042-1060  925-943 D-3719 >0.0488−0.6 >12.5 −0.20  526-544 — D-3720 >12.5 −0.2 >12.5 −0.10  523-541 —D-3721 >12.5 −0.2 >12.5 −0.10  694-712  577-595 D-3722 0.0051033−0.9 >12.5 −0.20 1168-1186 1051-1069 D-3723 >0.195 −0.5 >12.5 −0.20 891-909  774-792 D-3724 0.00819 −0.9 >12.5 −0.30 1052-1070  935-953D-3725 >12.5 −0.2 >12.5 −0.20 1116-1134   99-1017 D-3726 0.00546−0.5 >12.5 −0.50 1228-1246 1111-1129 D-3727 0.006855 −0.9 >12.5 −0.50 952-970  835-853 D-3728 >12.5 −0.2 >12.5 −0.30  975-993  858-876D-3729 >12.5 −0.1 >12.5 −0.20  850-868  733-751 D-3730 >12.5 −0.1 >3.13−0.20  288-306  288-306 D-3731 0.0106 −0.7 >12.5 −0.10 1034-1052 917-935 D-3732 4.07 −0.5 >12.5 0.20 1180-1198 1063-1081 D-3733 0.0311−0.4 >12.5 −0.20  897-915  780-798 D-3734 0.005955 −0.9 >12.5 −0.201045-1063  928-946 D-3735 >1.56 −0.6 >12.5 −0.40 1225-1243 1108-1126D-3736 >0.0488 −0.6 >12.5 −0.30 1073-1091  956-974 D-3737 0.086915−0.7 >12.5 −0.20 1260-1278 1143-1161 D-3738 0.68126 −0.9 >12.5 −0.10 796-814  679-697 D-3739 0.024703 −0.9 >12.5 −0.30  526-544 —D-3740 >12.5 −0.5 >12.5 −0.10  523-541 — D-3741 0.0982 −0.8 >12.5 −0.10 891-909  774-792 D-3742 0.07107 −0.6 >12.5 0.10  288-306  288-306D-3743 0.025805 −0.6 >12.5 −0.10  897-915  780-798 D-3744 0.03467−0.8 >12.5 0.10  888-906  771-789 D-3745 0.000851 −1.0 ND ND  692-710 575-593 D-3746 0.00158 −0.9 ND ND  692-710  575-593 D-3747 0.00289 −0.6ND ND  692-710  575-593 D-3748 0.0301 −0.6 ND ND  692-710  575-593D-3749 0.00133 −0.9 ND ND  692-710  575-593 D-3750 0.00327 −0.6 ND ND 692-710  575-593 D-3751 0.0043167 −1.0 0.0193 −0.70  692-710  575-593D-3752 0.00545 −0.9 0.0507 −0.50  692-710  575-593 D-3753 0.00918−1.0 >25.0 0.10  692-710  575-593 D-3754 0.0029367 −1.0 >25.0 0.10 692-710  575-593 D-3755 0.00034033 −0.9 >25.0 0.20  692-710  575-593D-3756 0.0276 −0.5 >25.0 0.10  692-710  575-593 D-3757 0.014369−1.0 >25.0 0.10  888-906  771-789 D-3758 0.0029492 −0.9 >0.5 −0.20 691-709  574-592 D-3759 0.022895 −0.8 >0.5 0.10  692-710  575-593D-3760 0.0010037 −0.8 ND ND  888-906  771-789 D-3761 >0.0156 −0.3 ND ND 692-710  575-593 D-3762 0.002224 −0.7 ND ND  692-710  575-593 D-37630.001353 −0.8 ND ND  692-710  575-593 D-3764 >0.5 0.1 ND ND  692-710 575-593 D-3765 0.0009435 −0.8 ND ND  692-710  575-593 D-3766 0.000828−0.7 ND ND  692-710  575-593 D-3767 0.0037575 −0.9 0.01547 −0.80 692-710  575-593 D-3768 0.024725 −0.9 0.0662 −0.60  692-710  575-593D-3769 0.00231 −0.4 0.0265 −0.20  692-710  575-593 D-3770 >0.5 −0.5 NDND  692-710  575-593 D-3771 0.001805 −0.8 ND ND  692-710  575-593 D-37720.001135 −0.9 ND ND  888-906  771-789 D-3773 0.003285 −1.0 ND ND 692-710  575-593 D-3774 0.004225 −1.0 0.0415 −0.60  692-710  575-593D-3775 0.003205 −0.9 0.03335 −0.50  888-906  771-789 D-3776 0.25585−0.7 >12.5 −0.20  888-906  771-789 D-3777 0.12353 −1.0 >12.5 −0.30 888-906  771-789 D-3778 0.00263 −0.76 ND ND 1185-1203 1068-1086D-3779 >0.391 −0.58 ND ND 1393-1411 1276-1294 D-3780 0.00496 −0.82 ND ND 395-413  395-413 D-3781 >0.195 −0.55 ND ND  396-414  396-414 D-37820.00437 −0.79 ND ND 1052-1070  935-953 D-3783 >0.0977 −0.39 ND ND1073-1091  956-974 D-3784 0.00799 −0.69 ND ND 1074-1092  957-975 D-37850.011 −0.66 ND ND 1116-1134   99-1017 D-3786 >12.5 −0.38 ND ND 1168-11861051-1069 D-3787 >12.5 −0.30 ND ND 1180-1198 1063-1081 D-3788 0.00817−0.70 ND ND 1425-1444 1308-1327 D-3789 0.00365 −0.48 ND ND 1341-13591224-1242 D-3790 >6.25 −0.74 ND ND 1424-1443 1307-1326 D-3791 0.00241−0.74 ND ND  991-1009  874-892 D-3792 >12.5 −0.35 ND ND 1081-1099 964-982 D-3793 >12.5 −0.27 ND ND 1164-1182 1047-1065 D-3794 >12.5 −0.46ND ND  563-581 — D-3795 0.00349 −0.77 ND ND 1225-1243 1108-1126D-3796 >0.781 −0.43 ND ND 1260-1278 1143-1161 D-3797 >0.781 −0.63 ND ND 772-790  655-673 D-3798 >0.781 −0.39 ND ND  886-904  769-787 D-37990.0108 −0.76 ND ND  891-909  774-792 D-3800 0.0039 −0.80 ND ND  897-915 780-798 D-3801 0.00575 −0.76 ND ND  888-906  771-789 D-3802 0.00406−0.95 >0.5 0.09  888-906  771-789 D-3803 0.01116 −0.91 >0.5 −0.08 888-906  771-789 D-3804 0.00477 −0.97 >0.5 −0.07  692-710  575-593D-3805 >0.5 0.07 >0.5 −0.08  692-710  575-593 D-3806 >0.5 0.13 >0.5 0.04 692-710  575-593 D-3807 >0.5 0.15 >0.5 −0.18  692-710  575-593 D-3813ND ND 0.02753 −0.69  692-710  575-593 D-3814 ND ND 0.00708 −0.70 692-710  575-593 D-3815 ND ND 0.00987 −0.49  692-710  575-593 D-3816 NDND No curve fit 0.074826181  692-710  575-593 D-3817 ND ND 0.02035 −0.65 692-710  575-593 D-3818 ND ND 0.02340 −0.68  692-710  575-593 D-3819 NDND 0.01316 −0.70  692-710  575-593 D-3820 ND ND 0.02094 −0.75  692-710 575-593 D-3821 ND ND 0.03345 −0.75  692-710  575-593 D-3822 ND ND0.03030 −0.65  692-710  575-593 D-3823 ND ND 0.05948 −0.72  692-710 575-593 D-3824 ND ND 0.01883 −0.43  692-710  575-593 D-3825 ND ND0.01320 −0.80  692-710  575-593 D-3826 ND ND 0.07031 −0.76  692-710 575-593 D-3827 ND ND 0.02455 −0.61 1186-1205 1069-1088 D-3828 ND ND Nocurve fit 0.07  170-189  170-189 D-3829 ND ND No curve fit 0.14  673-692 556-575 D-3830 ND ND No curve fit 0.18  668-687  551-570 D-3831 ND NDNo curve fit 0.188  696-715  579-598 D-3832 ND ND 0.01483 −0.78  168-187 168-187 D-3833 ND ND No curve fit 0.17 1075-1094  958-977 D-3834 ND NDNo curve fit 0.13 1084-1103  967-986 D-3835 ND ND No curve fit 0.20 157-176  157-176 D-3836 ND ND No curve fit −0.20 1423-1442 1306-1325D-3837 ND ND No curve fit 0.13  970-989  853-872 D-3838 ND ND No curvefit 0.16  992-1011  875-894 D-3839 ND ND No curve fit 0.17  171-190 171-190 D-3840 ND ND No curve fit −0.09  606-625  489-508 D-3841 ND NDNo curve fit 0.06  393-412  393-412 D-3842 ND ND No curve fit 0.080632-0651  515-534 D-3843 ND ND No curve fit 0.17 0990-1009  873-892D-3844 ND ND 0.11508 −0.50 1050-1069  933-952 D-3845 ND ND No curve fit−0.26  807-826  690-709 D-3846 ND ND No curve fit −0.05  166-185 166-185 D-3847 ND ND No curve fit 0.19 1083-1102  966-985 D-3848 ND ND0.01367 −0.41 1064-1083  947-966 D-3849 ND ND No curve fit 0.17  328-347 328-347 D-3850 ND ND No curve fit 0.12 1079-1098  962-981 D-3851 ND NDNo curve fit 0.09 1123-1142 1006-1025 D-3852 ND ND No curve fit −0.04 605-624  488-507 D-3853 ND ND No curve fit 0.22  175-194  175-194D-3854 ND ND No curve fit 0.29  692-710  575-593 D-3855 ND ND No curvefit 0.14  692-710  575-593 D-3856 ND ND No curve fit 0.11  692-710 575-593 D-3857 0.00532 −0.61 >0.5 −0.44

Several of the GalNAc-ASGR1 siRNA conjugates were evaluated further forefficacy in knocking down ASGR1 mRNA levels in hepatocytes. Followingthe manufacturers protocol, human primary hepatocyte cells(Xenotech/Sekisui donor lot #HC3-38) were thawed in OptiThaw media(Xenotech cat #K8000). Cells were centrifuged and post media aspiration,resuspended in OptiPlate hepatocyte media (Xenotech cat #K8200) andplated into 96 well collagen coated plates (Greiner cat #655950).Following a 2-4 hour incubation period, media was removed and replacedwith OptiCulture hepatocyte media (Xenotech cat #K8300). 2-4 hoursfollowing the addition of OptiCulture media, GalNAc-conjugated siRNAswere delivered to cells via free uptake (no transfection reagent). Cellswere incubated 24-72 hours at 37° C. and 5% CO₂. Cells were then lysedwith Qiagen RLT buffer (79216)+1% 2-mercaptoethanol (Sigma, M-3148), andthe lysates were stored at −20° C. RNA was purified using a QiagenQIACube HT instrument (9001793) and a Qiagen RNeasy 96 QIACube HT Kit(74171) according to manufacturer's instructions. Samples were analyzedusing a QIAxpert system (9002340).

cDNA was synthesized from RNA samples using the Applied Biosystems HighCapacity cDNA Reverse Transcription kit (4368813), reactions wereassembled according to manufacturer's instructions, input RNAconcentration varied by sample. Reverse transcription was carried out ona BioRad tetrad thermal cycler (model #PTC-0240G) under the followingconditions: 25° C. 10 minutes, 37° C. 120 minutes, 85° C. 5 minutesfollowed by (an optional) 4° C. infinite hold. Droplet digital PCR(ddPCR) was performed using BioRad's QX200 AutoDG droplet digital PCRsystem according to manufacturer's instructions. Reactions wereassembled into an Eppendorf clear 96 well PCR plate (951020303) usingBioRad ddPCR Supermix for Probes (1863010), fluorescently labeled qPCRassays for ASGR1 (IDT Hs.PT.56a.24725395, ordered with primer to proberatio 3.6:1, 9 nanomoles each forward and reverse primer (sequenceslisted below), 2.5 nanomoles 6-FAM/ZEN/IBFQ labeled probe (sequencelisted below)) and GUSB (IDT Hs.PT.58v.27737538, ordered with primer toprobe ratio 3.6:1, 9 nanomoles each forward and reverse primer(sequences listed below), 2.5 nanomoles HEX/ZEN/IBFQ labeled probe(sequence listed below)) and RNase free water (Ambion, AM9937). Finalprimer/probe concentration was 900 nM/250 nM respectively, input cDNAconcentration varied among wells.

Droplets were formed using a BioRad Auto DG droplet generator (1864101)set up with manufacturer recommended consumables (BioRad DG32 cartridges1864108, BioRad tips 1864121, Eppendorf blue 96 well PCR plate951020362, BioRad droplet generation oil for probes 1864110 and a BioRaddroplet plate assembly). Droplets were amplified on a BioRad C1000 touchthermal cycler (1851197) using the following conditions: enzymeactivation 95° C. 10 minutes, denaturation 94° C. 30 seconds followed byannealing/extension 60° C. for one minute, 40 cycles using a 2°C./second ramp rate, enzyme deactivation 98° C. 10 minutes followed by(an optional) 4° C. infinite hold. Samples were then read on a BioRadQX200 Droplet Reader measuring FAM/HEX signal that correlated to ASGR1or GUSB concentration, respectively. Data was analyzed using BioRad'sQuantaSoft software package. Samples were gated by channel (fluorescentlabel) to determine the concentration per sample. Each sample was thenexpressed as the ratio of the concentration of the gene of interest(ASGR1)/concentration of the housekeeping gene (GUSB) to control fordifferences in sample loading. Data was then imported into GenedataScreener, where each test siRNA was normalized to the median of theneutral control wells (buffer only) and was expressed as the POC(percent of control). IC50 and max activity are reported in Table 10below.

ddPCR Assay Sequences ASGR1: Primer 1: (SEQ ID NO: 4688)CAGGCTGGAGTGATCTTCA Primer 2: (SEQ ID NO: 4689) TTCAGCAACTTCACAGCGAProbe: (SEQ ID NO: 4690) 56-FAM/TCTTTCTTC   (SEQ ID NO: 4691)/ZEN/CCACATTGCCTCCCTG/3IABkFQ/ GUSB: Primer 1: (SEQ ID NO: 4692)GTTTTTGATCCAGACCCAGATG Primer 2: (SEQ ID NO: 4693) GCCCATTATTCAGAGCGAGTAProbe: (SEQ ID NO: 4694) 5HEX/TGCAGGGTT   (SEQ ID NO: 4695)/ZEN/TCACCAGGATCCAC/3IABkFQ/

TABLE 10 ASGR1 mRNA in vitro knockdown by select GalNAc-ASGR1 conjugatesPrimary Human Hepatocyte Primary Human Hepatocyte Duplex No. ddPCR IC50(μM) ddPCR Max Activity D-3779 0.0302 −0.46 D-3780 0.0040 −0.80 D-37810.0158 −0.40 D-3782 0.0084 −0.79 D-3783 — −0.19 D-3784 0.0281 −0.42D-3785 0.0249 −0.40 D-3786 0.0267 −0.32 D-3787 — 0.03 D-3788 0.0127−0.49 D-3789 — −0.26 D-3790 0.0046 −0.34 D-3791 0.0041 −0.81 D-3792 —−0.03 D-3793 — −0.09 D-3794 — −0.05 D-3795 0.0022 −0.66 D-3796 — 0.17D-3797 0.0875 −0.45 D-3798 0.0114 −0.38 D-3799 0.0135 −0.36 D-38000.0094 −0.73 D-3801 0.0159 −0.57

The majority of the tested GalNAc-siRNA conjugates reduced ASGR1 mRNAlevels in primary human hepatocytes indicating that the conjugates wereeffectively delivered to the cells and the siRNAs were active. CompoundsD-3780, D-3782, D-3791, D-3795, and D-3800 were the most potent and hadthe highest maximum inhibitory activity of the conjugates evaluated inthis assay. These compounds also exhibited potent inhibition of ASGR1protein expression when transfected into Hep3B cells. See Hep3Btransfection assay data in Table 9.

Example 9. In Vivo Efficacy of GalNAc-ASGR1 siRNA Conjugates

To assess whether the GalNAc-ASGR1 siRNA conjugates could effectivelysilence ASGR1 expression in vivo, conjugates exhibiting the best invitro inhibition as measured by ddPCR (Table 10) were administered toASGR1 knockout mice expressing the human ASGR1 gene. 10-12 week oldASGR1 knockout mice (The Jackson Laboratory) were i.v. injected with anadeno-associated virus (AAV) encoding the human ASGR1 gene (AAV-hASGR1)at a dose of 1×10¹² genome copies (GC) per animal. Two weeks followingAAV-hASGR1 injection, mice received a s.c. injection of buffer or theindicated GalNAc-siRNA conjugate (compounds D-3752, D-3779, D-3780,D-3782, D-3784, D-3785, D-3788, D-3791, D-3795, D-3797, D-3799, D-3800,and D-3801) at 5 mg/kg body weight in 0.25 ml buffer (n=6 each group).At day 8 following compound administration, three animals in eachtreatment group were euthanized and harvested for further analysis. Theremaining three animals in each treatment group were harvestred at day15 following compound administration. Serum and livers were collectedfrom all animals. Total RNA isolated from the livers of the animals wasprocessed for qPCR analysis to assess human ASGR1 mRNA levels. Serumlevels of alkaline phosphatase (ALP) were measured by a clinicalanalyzer (AU400 Chemistry Analyzer, Olympus). Elevated levels of ALP inthe serum has been reported to correlate with reduced serum levels ofnon-HDL cholesterol and reduced risk of coronary artery disease (Nioi etal., New England Journal of Medicine, Vol. 374(22):2131-2141, 2016), andthus serves as useful biomarker.

As shown in FIG. 6A, several of the GalNAc-siRNA conjugates reducedhuman ASGR1 mRNA levels 8 days following administration. CompoundsD-3752, D-3779, D-3782, D-3788, D-3799, and D-3800 were particularlyeffective. Suppression of hASGR1 mRNA levels 15 days following compoundadministration was observed for some of the compounds. Compounds D-3752and D-3788 were especially effective at this time point. FIG. 6B showsserum ALP levels at the same time points. Generally, elevation of serumALP levels correlated with knockdown of hASGR1 mRNA. Some of thecompounds (e.g. D-3752, D-3782) produced elevation of serum ALP tolevels similar to those observed in ASGR1 knockout animals, whichrepresent maximum inhibition of ASGR1 expression. The results of the invivo experiments demonstrate that the GalNAc-ASGR1 siRNA conjugates,when administered subcutaneously, effectively suppress ASGR1 geneexpression in the liver and modulate serum ALP, a biomarker of efficacyin treating coronary artery disease.

Example 10. ASGR1 Antibody as an Alternative Delivery Mechanism forsiRNA Molecules

The purpose of the experiments described in this example was todetermine whether a monoclonal antibody against ASGR1 could be used todeliver ASGR1 siRNA molecules to the liver. An anti-ASGR1 monoclonalantibody with an E272C mutation in its heavy chain according to the EUnumbering scheme (anti-ASGR1 cys mAb, 200 mg) was incubated with 50 mLsolution of 2.5 mM cystamine and 2.5 mM cysteamine in 40 mM HEPESbuffer, pH 7.5-8.5 for 15-20 h at RT. The amino acid sequences of theheavy chain and light chain of the anti-ASGR1 antibody are providedbelow as SEQ ID NOs: 4696 and 4697, respectively. The reaction mixturewas filtered using a 0.22 μm filter, and diluted to 250 mL with 100 mMsodium acetate buffer pH 5. Cation exchange chromatography was performedto purify the bis-cysteamine-capped anti-ASGR1 cys mAb from the reactionmixture. First, 250 mL of reaction mixture diluted in 100 mM sodiumacetate buffer pH 5 was loaded onto 25 mL SP HP column (GE HealthcareLife Sciences) at 5 mL/min. The column was washed with 2 column volumes(CV) of 100 mM sodium acetate pH 5, followed by a 0-20% gradient of 100mM sodium acetate with 1.2 M sodium chloride (NaCl) pH 5 over 10 CV. Themain peak containing bis-cysteamine-capped anti-ASGR1 cys mAb wascollected and buffer exchanged into 10 mM sodium acetate with 9% sucrosepH 5.2 via dialysis.

Anti-ASGR1 Cys mAb Heavy Chain (SEQ ID NO: 4696)QVQLVESGGGVVQPGRSLRLSCAASGFTESSYGMEIWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRETISRDNSKNTLYLQMNSLRAEDTAVYYCARDSSPYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPCVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAnti-ASGR1 Cys mAb Light Chain (SEQ ID NO: 4697)DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKWYGASSLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQSDSFPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

A siRNA duplex containing a sense strand having a sequence (5′ to 3′) ofGfsusGfgGfaAfgAfAfAfgAfuGfaAfgUfuUf (SEQ ID NO: 4698) and an antisensestrand having a sequence (5′ to 3′) ofasCfsuUfcAfuCfuuuCfuUfcCfcAfcsUfsu (SEQ ID NO: 4699) was used togenerate the mAb-siRNA conjugate. The notations in the sense andantisense sequences are the same as those used for the nucleotidesequences in Tables 6 and 8 described above. The siRNA duplex had a 19base pair duplex region with a 2 nucleotide overhang at the 3′ end ofthe sense and antisense strands. The sense strand of the siRNA duplexhad a homoserine-aminohexanoic acid (hSer-Ahx) modification at its 3′end. The siRNA duplex was formed in 100 mM potassium acetate, 30 mMHEPES-KOH, pH 7.4 upon heating to 90° C. for 5 min and cooling to RTover 30 min. The 3′ hSer-Ahx siRNA duplex was further functionalizedwith a bromoacetyl group using succinimidyl bromoacetate (SBA) (FIG.7A). The 3′ hSer-Ahx siRNA duplex in 100 mM potassium acetate, 30 mMHEPES-KOH, pH 7.4 was incubated with 10-20 equivalents of SBA at RT for1 h. Then, an additional 10-20 equivalents of SBA were added, and thereaction mixture was incubated at RT for another hour. The reaction wasmonitored using LC-TOF. Excess SBA was removed from the3′-bromoacetyl-siRNA by buffer exchanging with 50 mM sodium phosphate, 2mM ethylenediaminetetraacetic acid (EDTA), pH 7.5 using Amicon-15 3kspin concentrators.

Bis-cysteamine-capped anti-ASGR1 cys mAb (˜5 mg/mL in 10 mM sodiumacetate with 9% sucrose) was partially reduced using 3-4 equivalents oftris(2-carboxyethyl)phosphine (TCEP) ortriphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt (TPPTS) at RTfor 60-90 min (FIG. 7B). The reaction was monitored using analyticalcation exchange chromatography. TCEP or TPPTS was removed, and partiallyreduced cys mAb was buffer exchanged into 50 mM sodium phosphate bufferpH 7.5 containing 2 mM EDTA. To the partially reduced cys mAb was added6-10 equivalents of dehydroascorbic acid (DHAA), and oxidation wascarried out at RT until only trace amount of reduced mAb species wereobserved (30-180 min). Without removing DHAA, 6 equivalents ofbromoacetyl-siRNA duplex were added to the reaction mixture, and thealkylation was carried out at RT for 15-48 h (FIG. 7B). Excess siRNAduplex and small molecule reagents were removed by size exclusionchromatography (SEC) with isocratic flow of 0.17 M potassium phosphate,0.21 M potassium chloride, 10% (v/v) isopropanol, pH 7. The anti-ASGR1mAb-siRNA conjugates with RNA-to-antibody ratio (RAR) of 1 and 2 wereseparated using anion exchange chromatography. The SEC pool was dilutedin 20 mM Tris-HCl pH 7, 100 mM NaCl and loaded onto Q HP column (GEHealthcare Life Sciences). The column was washed with 5 CV of 20 mMTris-HCl pH 7, 100 mM NaCl, followed by a gradient elution with 20 mMTris-HCl pH 7 containing 0.4 to 1 M NaCl over 20 CV. The purified RAR1(compound 3549) and RAR2 (compound 3550) products were buffer exchangedinto Dulbecco's phosphate-buffered saline (DPBS) using spinconcentration.

The mAb-siRNA conjugates were evaluated for activity in a free uptakeassay to determine whether the antibody could effectively deliver thesiRNA to human primary hepatocytes to inhibit ASGR1 expression. Variousconcentrations (0.18 nM to 400 nM) of the anti-ASGR1 mAb conjugated to 1or 2 ASGR1 siRNA molecules (compounds 3549 and 3550, respectively) wereincubated with human primary hepatocytes for four days. RNA was isolatedfrom the cells and processed for droplet digital PCR analysis to assessASGR1 mRNA levels as described in Example 8. The results of the in vitroassay are shown in FIG. 8. The anti-ASGR1 mAb conjugated to 1 or 2 ASGR1siRNA molecules demonstrated 40-60% knockdown of ASGR1 mRNA. Theunconjugated anti-ASGR1 cys mAb (PL-53515) was used as a control.

Next, the anti-ASGR1 mAb-siRNA conjugates were tested for in vivoefficacy. Nine-week old C57Bl/6 wild-type mice were injectedsubcutaneously or intravenously with compound 3550 (30 mg/kg or 60mg/kg) or a GalNAc-conjugated siRNA control. The GalNAc-conjugated siRNAcontrol had a sense strand having a sequence of SEQ ID NO: 4698 and anantisense strand having a sequence of SEQ ID NO: 4699 and was conjugatedto a triantennary GalNAc moiety at the 3′ end of the sense strand. Serumand livers were collected from the animals at days 2, 4, 8, and 15following compound administration. Total RNA isolated from the livers ofthe animals was processed for qPCR analysis to assess ASGR1 mRNA levels.ASGR1 protein expression in the liver was measured by ELISA. Serumlevels of alkaline phosphatase (ALP) were measured by a clinicalanalyzer (AU400 Chemistry Analyzer, Olympus).

The mAb-siRNA conjugate 3550 effectively delivered siRNA to its mRNAtarget in vivo. The highest knockdown level (˜80%) resulted from 30 mpki.v. administration of 3550 in wild-type mice measured on day 8 (FIG.9A). The ASGR1 protein expression in liver was also measured, and >80%reduction in ASGR1 protein was achieved in the 30 mpk i.v. group,consistent with the level of mRNA knockdown (FIG. 9B). Nadir of proteinknockdown was day 8 for the anti-ASGR1 mAb-siRNA conjugate, dosed eitheri.v. or s.c., and day 4 for the GalNAc-siRNA conjugate. The mAb-siRNAconjugate 3550 resulted in 2-4 fold increase in ALP on day 8,corresponding to decreased ASGR1 mRNA level and protein expression (FIG.10). The anti-ASGR1 antibody alone did not induce any increase in ALPeven at 100 mg/kg (data not shown).

Taken together, the results of these experiments demonstrate that siRNAduplexes can be effectively delivered to the liver using an anti-ASGR1antibody in lieu of a GalNAc moiety. The mAb-siRNA conjugates exhibitedcomparable efficacy to a GalNAc-siRNA conjugate in terms of inhibitionof liver ASGR1 expression and elevation of serum ALP levels, a biomarkerof target inhibition.

All publications, patents, and patent applications discussed and citedherein are hereby incorporated by reference in their entireties. It isunderstood that the disclosed invention is not limited to the particularmethodology, protocols and materials described as these can vary. It isalso understood that the terminology used herein is for the purposes ofdescribing particular embodiments only and is not intended to limit thescope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An RNAi construct comprising a sense strand and an antisense strand,wherein the antisense strand comprises a region having a sequence thatis complementary to an ASGR1 mRNA sequence, and wherein said regioncomprises at least 15 contiguous nucleotides from an antisense sequenceselected from any one of SEQ ID NOs: 1508-3010.
 2. The RNAi construct ofclaim 1, wherein the sense strand comprises a sequence that issufficiently complementary to the sequence of the antisense strand toform a duplex region of about 15 to about 30 base pairs in length. 3.(canceled)
 4. (canceled)
 5. The RNAi construct of claim 1, wherein thesense strand and the antisense strand are each about 15 to about 30nucleotides in length.
 6. The RNAi construct of claim 5, wherein thesense strand and the antisense strand are each about 19 to about 27nucleotides in length.
 7. The RNAi construct of claim 5, wherein thesense strand and the antisense strand are each about 21 to about 25nucleotides in length.
 8. The RNAi construct of claim 1, wherein theRNAi construct comprises at least one blunt end.
 9. The RNAi constructof claim 1, wherein the RNAi construct comprises at least one nucleotideoverhang of 1 to 4 unpaired nucleotides.
 10. (canceled)
 11. The RNAiconstruct of claim 9, wherein the RNAi construct comprises a nucleotideoverhang at the 3′ end of the sense strand, the 3′ end of the antisensestrand, or the 3′ end of both the sense strand and the antisense strand.12. (canceled)
 13. The RNAi construct of claim 1, wherein the RNAiconstruct comprises at least one modified nucleotide.
 14. The RNAiconstruct of claim 13, wherein the modified nucleotide is a 2′-modifiednucleotide.
 15. The RNAi construct of claim 13, wherein the modifiednucleotide is a 2′-fluoro modified nucleotide, a 2′-O-methyl modifiednucleotide, a 2′-O-methoxyethyl modified nucleotide, a 2′-O-allylmodified nucleotide, a bicyclic nucleic acid (BNA), or combinationsthereof.
 16. (canceled)
 17. The RNAi construct of claim 13, wherein allof the nucleotides in the sense and antisense strands are modifiednucleotides.
 18. The RNAi construct of claim 17, wherein the modifiednucleotides are 2′-O-methyl modified nucleotides, 2′-fluoro modifiednucleotides, or combinations thereof.
 19. The RNAi construct of claim 1,wherein the RNAi construct comprises at least one phosphorothioateinternucleotide linkage.
 20. The RNAi construct of claim 19, wherein theRNAi construct comprises two consecutive phosphorothioateinternucleotide linkages at the 3′ end of the antisense strand.
 21. TheRNAi construct of claim 19, wherein the RNAi construct comprises twoconsecutive phosphorothioate internucleotide linkages at both the 3′ and5′ ends of the antisense strand and two consecutive phosphorothioateinternucleotide linkages at the 5′ end of the sense strand.
 22. The RNAiconstruct of claim 1, wherein the antisense strand comprises a sequenceselected from any one of SEQ ID NOs: 1508-3010.
 23. (canceled)
 24. TheRNAi construct of claim 22, wherein the sense strand comprises asequence selected from any one of SEQ ID NOs: 5-1507.
 25. (canceled) 26.The RNAi construct of claim 22, wherein: (a) the sense strand comprisesthe sequence of SEQ ID NO: 181 and the antisense strand comprises thesequence of SEQ ID NO: 1684; (b) the sense strand comprises the sequenceof SEQ ID NO: 205 and the antisense strand comprises the sequence of SEQID NO: 1708; (c) the sense strand comprises the sequence of SEQ ID NO:211 and the antisense strand comprises the sequence of SEQ ID NO: 1714;(d) the sense strand comprises the sequence of SEQ ID NO: 240 and theantisense strand comprises the sequence of SEQ ID NO: 1743; (e) thesense strand comprises the sequence of SEQ ID NO: 820 and the antisensestrand comprises the sequence of SEQ ID NO: 2323; (f) the sense strandcomprises the sequence of SEQ ID NO: 1147 and the antisense strandcomprises the sequence of SEQ ID NO: 2650; (g) the sense strandcomprises the sequence of SEQ ID NO: 1148 and the antisense strandcomprises the sequence of SEQ ID NO: 2651; (h) the sense strandcomprises the sequence of SEQ ID NO: 1364 and the antisense strandcomprises the sequence of SEQ ID NO: 2867; (i) the sense strandcomprises the sequence of SEQ ID NO: 1366 and the antisense strandcomprises the sequence of SEQ ID NO: 2869; or (j) the sense strandcomprises the sequence of SEQ ID NO: 1370 and the antisense strandcomprises the sequence of SEQ ID NO:
 2873. 27-34. (canceled)
 35. TheRNAi construct of claim 1, wherein the RNAi construct further comprisesa ligand.
 36. The RNAi construct of claim 35, wherein the ligandcomprises a cholesterol moiety, a vitamin, a steroid, a bile acid, afolate moiety, a fatty acid, a carbohydrate, a glycoside, or antibody orantigen-binding fragment thereof.
 37. The RNAi construct of claim 35,wherein the ligand targets delivery of the RNAi construct tohepatocytes.
 38. The RNAi construct of claim 37, wherein the ligandcomprises a monoclonal antibody or antigen-binding fragment thereof thatspecifically binds to human ASGR1.
 39. The RNAi construct of claim 38,wherein the monoclonal antibody or antigen-binding fragment thereofcomprises a substitution of at least one amino acid with a cysteineamino acid, and wherein the sense strand is covalently attached to themonoclonal antibody or antigen-binding fragment thereof through the sidechain of the cysteine amino acid.
 40. The RNAi construct of claim 35,wherein the ligand comprises galactose, galactosamine, orN-acetyl-galactosamine.
 41. The RNAi construct of claim 40, wherein theligand comprises a multivalent galactose moiety or multivalentN-acetyl-galactosamine moiety.
 42. The RNAi construct of claim 41,wherein the multivalent galactose moiety or multivalentN-acetyl-galactosamine moiety is trivalent or tetravalent.
 43. The RNAiconstruct of claim 35, wherein the ligand is covalently attached to thesense strand optionally through a linker.
 44. The RNAi construct ofclaim 43, wherein the ligand is covalently attached to the 3′ end or 5′end of the sense strand.
 45. A pharmaceutical composition comprising theRNAi construct of claim 1 and a pharmaceutically acceptable carrier,excipient, or diluent.
 46. A method for reducing the expression of ASGR1in a patient in need thereof comprising administering to the patient theRNAi construct of claim
 1. 47-50. (canceled)
 51. A method for reducingnon-HDL cholesterol in a patient in need thereof comprisingadministering to the patient the RNAi construct of claim
 1. 52.(canceled)
 53. A method for treating or preventing cardiovasculardisease in a patient in need thereof comprising administering to thepatient the RNAi construct of claim
 1. 54. (canceled)
 55. A method forreducing the risk of myocardial infarction in a patient in need thereofcomprising administering to the patient the RNAi construct of claim 1.56-73. (canceled)